Therapeutic apoptotic cell preparations, method for producing same and uses thereof

ABSTRACT

The present application provides pharmaceutical compositions comprising a population of mononuclear-enriched cells in an early-apoptotic state, methods for the production of said compositions and uses thereof in the treatment of diseases characterized by pathological immune responses. The pharmaceutical compositions may be used in treatment of conditions such as, but not limited to, graft versus host disease (GVHD) and autoimmune diseases including but not limited to inflammatory bowel disease, gout and arthritis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Phase Application of PCT InternationalApplication No. PCT/IL2013/051001, International Filing Date Dec. 5,2013, claiming priority of U.S. Provisional Patent Applications Nos.61/733,936, filed Dec. 6, 2012; 61/778,497, filed Mar. 13, 2013; and61/872,884, filed Sep. 3, 2013, which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a therapeutic cell populationcomprising apoptotic cells and, more particularly, to compositionscomprising mononuclear enriched cells in an early apoptotic state,methods for the production thereof, and uses thereof in treatment ofdiseases characterized by pathological immune responses.

BACKGROUND OF THE INVENTION

Diseases characterized by pathological immune responses include manydiseases associated with significant mortality and morbidity,particularly autoimmune diseases, such as systemic lupus erythematosus(SLE), and transplantation-related diseases such as graft-versus-hostdisease (GVHD). Autoimmune diseases may generally be divided into twogeneral types, namely systemic autoimmune diseases (e.g. SLE andscleroderma), and organ specific autoimmune diseases, such as multiplesclerosis, and diabetes.

Immunosuppressive drugs have been used for treatment or prevention ofthe rejection of transplanted organs and tissues (e.g., bone marrow,heart, kidney, liver); for treatment of autoimmune diseases or diseasesthat are most likely of autoimmune origin (e.g., rheumatoid arthritis,multiple sclerosis, myasthenia gravis, systemic lupus erythematosus,sarcoidosis, Crohn's disease, Behcet's Disease, pemphigus, uveitis andulcerative colitis); treatment of some other non-autoimmune inflammatorydiseases (e.g., long term allergic asthma control) as well astransplantation-related diseases (e.g. GVHD). However, immunosuppressivedrug treatments can lead to many complications, and improved methods fordealing with pathological immune reactions are needed.

Approximately 30,000 patients annually in the U.S. and Europe undergoallogeneic bone marrow transplantation (BMT). In allogeneic bone marrowtransplantation (alloBMT), the infusion of donor marrow into thepatient's body entails the interaction of cells from two immune systems.Conditioning regimens for patients receiving allogeneic transplantsallow the donor stem cells to engraft in the patient by suppressing theimmune system. Once the donor's immune cells are established in thepatient's body, they may recognize the patient's own tissue and cells,including any residual cancer cells, as being different or foreign. Theimmune system may then cause damage to certain organs such the liver,gastrointestinal tract or skin; this effect is known asgraft-versus-host disease (GVHD).

As of today, GVHD prophylaxis comprises the combination ofimmunosuppressive drugs including a calcineurin inhibitor (CNI),cyclosporine or tacrolimus, and either methotrexate, mycophenolatemofetil (MMF), or sirolimus. However, acute GVHD still occurs in 35% to70% of BMT patients who receive transplants from human leukocyte antigen(HLA)-matched siblings, and even more frequently in unrelated donortransplant recipients.

Although calcineurin inhibitors (CNIs) partially inhibit acute GVHD,they may impair immune reconstitution by inhibiting T-cell developmentand increasing the risk of disease relapse. Thus, patients withhematologic malignancies undergoing allogeneic BMT are in need of GVHDprophylaxis that would minimize the use of CNIs, prevent GVHD, andretain a functional immune system including a beneficialgraft-versus-tumor effect.

U.S. Pat. Nos. 6,524,865, 6,607,722 and 7,109,031, and US patentapplications 2010/0267137, 2010/01837365 relate to the production ofimmunosuppressive recipient dendritic cells, and contacting thedendritic cells with necrotic or apoptotic donor leukocytes, intended toreduce immune response to grafts or implants.

WO 2002/060376, to one of the inventors of the present application,discloses a method of treatment of a systemic autoimmune disease in asubject by administration of apoptotic and/or necrotic cells obtainedfrom said subject.

WO 2006/117786, to one of the inventors of the present application,further discloses the use of a cell-preparation comprising dying or deadleukocytes for treatment of a disease characterized by a pathologicalimmune response. The dying or dead leukocytes are obtained by inducinglive leukocytes to adhere to a surface, and are capable of suppressingthe pathological immune response in the subject.

A study by Mevorach et al., published after the priority date of thepresent application, examined infusion of donor mononuclear earlyapoptotic cells as prophylaxis for acute graft-versus-host disease(GVHD) after HLA-matched myeloablative allogeneic hematopoietic stemcell transplantation (HSCT) from a related donor (Mevorach et al., ePubOctober 2013, Biol Blood Marrow Transplant).

During blood cell collection the use of anticoagulants is routine.During cell storage the use of anticoagulants has been reported toimprove cell yield. Matsumoto et al. compared storage of peripheralblood stem cells (PBSC) under various conditions, including storage inUniversity of Wisconsin (UW) solution and hypothermic preservation inautologous serum and the anticoagulant solution acid-citrate-dextrose(ACD) solution A. The survival of colony-forming unitgranulocyte-macrophages (CFU-GM) was found to be significantly better inUW solution than the survival achieved with hypothermic preservation inautologous serum and ACD-A solution at 4° C. or cryopreservation at 80°C. (Matsumoto et al., 2002, Bone Marrow Transplantation,30(11):777-784). Burger et al. disclosed that addition of heparin toplasma collected for cryopreservation of cells or addition of ACD-Aprevented gelation of freezing solution (Burger, S. R. et al, 1996,Transfusion, 36: 798-801). WO 2003/006691 discloses a cellcryopreservation medium for CD34+ cells comprising heparin. Kao et. al.discloses storage of bone marrow cell, peripheral blood stem cell orperipheral blood mononuclear cell products at 4° C. or 20° C. in mediacomprising ACD-A and/or heparin (Kao et al., 2011, Transfusion, 51:137-147). U.S. Pat. No. 6,489,311 discloses use of anticoagulants toprevent cell apoptosis.

There remains an unmet need for compositions and methods for treating orpreventing immune disorders including autoimmune and inflammatorydiseases and transplantation related diseases. For instance, GVHD, withan estimated incidence of 30%-70%, remains the main barrier forsuccessful allogeneic blood or marrow transplantation, and the optimalapproach for GVHD prophylaxis has not yet been established. Inparticular it is essential to obtain compositions and methods thatprevent or ameliorate GVHD in a safe, reliable, reproducible andeffective manner.

SUMMARY OF THE INVENTION

The present invention relates to a therapeutic population of earlyapoptotic cells. In particular, the present invention provides welldefined preparations of therapeutic mononuclear enriched cells at anearly-apoptotic state, improved methods for the production thereof, andthe use thereof in a clinical setting in treatment of diseasescharacterized by pathological immune responses. Examples of suchdiseases include, but are not limited to, graft versus host disease(GVHD), Crohn's disease and ulcerative colitis. In addition, the presentinvention provides methods to obtain therapeutic compositions comprisingmononuclear enriched early stage apoptotic cells with a stable andreproducible cell yield, and uses thereof.

The present invention is based, in part, on the finding thatmononuclear-enriched early-state apoptotic cells administered in aseparate infusion, in addition to the transplantation of bone marrowcells, have an ameliorative or prophylactic effect on GVHD. Inparticular, infusion of the apoptotic cell preparation of the inventionto subjects suffering from hematological malignancies who receivehematopoietic stem-cell transplantation (HSCT), was effective inreducing the incidence of acute high grade GVHD (e.g., grade II-IV). Inaddition, the apoptotic cell preparation remarkably reduced theincidence of hepatotoxicity in said subjects and was found in someinstances to reduce the time to engraftment of the HSCT.

The present invention is also based, in part, on the finding that asingle infusion of the apoptotic cell composition of the inventionsignificantly ameliorated both the clinical score and histologicaldamage in two different animal models of IBD.

During reduction to practice of the present invention in a clinicalsetting, the inventors encountered a problem in that producing the cellpreparation of the invention from blood of certain donors results in alow and/or unstable cell yield between different preparations. In someinstances cell yield was adversely affected by formation of cellaggregates in the resulting composition. This problem was especiallyprevalent in compositions produced using cells from donors having highlevels of blood triglycerides. In order to overcome these problems, oflow cell yield and/or formation of aggregates, it was found that use ofan anticoagulant during one or more stages of induction of apoptosis (inaddition to anticoagulant routinely used during cell collection) resultsin a high and stable cell yield in the composition. Furthermore,addition of anticoagulant during one or more stages of induction ofapoptosis enabled maintenance of a high and stable cell yield withindifferent preparations of the composition of the invention, regardlessof the protocol used for cell collection. According to some embodiments,as exemplified herein below, addition of anticoagulant during one ormore stages of induction of apoptosis enables production of acomposition with a high and stable cell yield of at least 30%,preferably at least 40%, typically at least 50% cells of the initialcells subjected to apoptosis induction. Each possibility represents aseparate embodiment of the present invention.

As exemplified herein below, such a high and stable cell yield isobserved both when producing the cell composition in the presence ofhigh or normal triglyceride levels. According to some embodiments, useof an anticoagulant during one or more stages of production of the cellcomposition of the invention results in a high and stable cell yield ofviable cells in the composition and/or of cells in an early-apoptoticstage in the composition. Each possibility represents a separateembodiment of the present invention. Thus, the present inventionprovides an early-apoptotic, stable and highly viablemononuclear-enriched cell composition, improved methods of producingsaid cell composition and use thereof in treating or amelioratingautoimmune and inflammatory diseases.

According to one aspect, the present invention provides a cellpreparation comprising mononuclear-enriched cells, wherein thepreparation comprises at least 85% mononuclear cells, wherein at least40% of the cells in the preparation are in an early-apoptotic state,wherein at least 85% of the cells in the preparation are viable cellsand wherein the preparation comprises no more than 15% CD15^(high)expressing cells.

According to another aspect, the present invention provides acomposition comprising the cell preparation of the invention. Accordingto some embodiments a composition comprising said cell preparationfurther comprises an anti-coagulant. According to some embodiments theanti-coagulant is selected from heparin and ACD Formula A orcombinations thereof. Each possibility represents a separate embodimentof the present invention. According to some embodiments, the heparin inthe composition comprising the final suspension medium used foradministration of said cell preparation to a patient is present at aconcentration between 0.005 U/ml and 2.5 U/ml. According to somealternative embodiments, the heparin in the composition comprising thefinal suspension medium used for administration of said cell preparationto a patient is present at a concentration between 0.01 U/ml and 1 U/ml.According to some embodiments, the ACD Formula A in the compositioncomprising the final suspension medium used for administration of saidcell preparation to a patient is present at a concentration of 0.01%-10%v/v. According to other embodiments, the ACD Formula A in thecomposition comprising the final suspension medium used foradministration of said cell preparation to a patient is present at aconcentration of 0.05%-5% v/v.

According to some embodiments, the composition of the invention furthercomprises residual methylprednisolone at a concentration that does notexceed 30 μg/ml. According to some embodiments, the composition of theinvention further comprises no more than 10% CD15^(high) expressingcells.

According to some embodiments, the cells in the cell-preparation of theinvention are collected from an allogeneic donor. According to someembodiments, the cells in the cell-preparation of the invention arecollected from the same donor as a donor of Hematopoietic Stem Cells(HSCs) used for a Hematopoietic Stem Cell Transplantation (HSCT)procedure. According to a non-limiting example, collection of cells froma donor is effected by leukapheresis. According to certain embodimentsthe mononuclear enriched cell preparation will contain autologous cells.

It is to be noted that, as known in the art, anti-coagulants areregularly used during cell-collection procedures, such as, but notlimited to, leukapheresis. According to some embodiments of the presentinvention, anticoagulant is further added to at least one medium usedduring preparation of the composition of the invention. According tosome embodiments, the at least one medium used during preparation of thecomposition of the invention is selected from the group consisting of:the freezing medium, the washing medium, the apoptosis inducingincubation medium and combinations thereof. Each possibility representsa separate embodiment of the present invention. According to someembodiments, the anti-coagulant is selected from the group consistingof: Heparin, ACD Formula A and a combination thereof. Each possibilityrepresents a separate embodiment of the present invention. It is to benoted that other anti-coagulants known in the art may be used accordingto the present invention, such as, but not limited to Fondaparinaux,Bivalirudin and Argatroban.

According to some embodiments, at least one medium used duringpreparation of the composition of the invention contains 5% of ACDformula A solution comprising 10 U/ml heparin. According to a typicalembodiment, anti-coagulant is not added to the final suspension mediumof the cell composition of the invention. As used herein, the terms“final suspension medium” and “administration medium” are usedinterchangeably.

According to some embodiments, at least one medium used duringpreparation of the composition of the invention comprises heparin at aconcentration of between 0.1-2.5 U/ml. According to some embodiments, atleast one medium used during preparation of the composition of theinvention comprises ACD Formula A at a concentration of between 1%-15%v/v. According to some embodiments, the freezing medium comprises ananti-coagulant. According to other embodiments, the incubation mediumcomprises an anti-coagulant. According to preferred embodiments, boththe freezing medium and incubation medium comprise an anti-coagulant.According to some embodiments the anti-coagulant is selected from thegroup consisting of: heparin, ACD Formula A and a combination thereof.Each possibility represents a separate embodiment of the presentinvention.

According to some embodiments, the heparin in the freezing medium is ata concentration of between 0.1-2.5 U/ml. According to some embodiments,the ACD Formula A in the freezing medium is at a concentration ofbetween 1%-15% v/v. According to some embodiments, the heparin in theincubation medium is at a concentration of between 0.1-2.5 U/ml.According to some embodiments, the ACD Formula A in the incubationmedium is at a concentration of between 1%-15% v/v. According tospecific embodiments, the anticoagulant is a solution ofacid-citrate-dextrose (ACD) formula A. In additional embodiments,exemplified herein below, the anticoagulant added to at least one mediumused during preparation of the composition of the invention is ACDFormula A containing heparin at a concentration of 10 U/ml.

In some embodiments, the mononuclear enriched cell preparation of theinvention comprises at least 85% mononuclear cells, preferably at least90% mononuclear cells. Each possibility is a separate embodiment of theinvention. According to some embodiments, the cell preparation comprisesat least 90% mononuclear cells. According to some embodiments, the cellpreparation comprises at least 95% mononuclear cells.

In additional embodiments, the mononuclear enriched cell preparationcomprises cell types selected from the group consisting of: lymphocytes,monocytes and natural killer cells. In another embodiment, themononuclear enriched cell preparation comprises no more than 15%,alternatively no more than 10%, typically no more than 5%polymorphonuclear leukocytes, also known as granulocytes (i.e.,neutrophils, basophils and eosinophils). Each possibility represents aseparate embodiment of the present invention. In yet another embodiment,the mononuclear enriched cell preparation comprises no more than 15%,alternatively no more than 10%, typically no more than 5% CD15^(high)expressing cells. Each possibility represents a separate embodiment ofthe present invention.

According to another aspect, the present invention provides a method forproducing the composition of the invention, the method comprising:

obtaining a mononuclear-enriched cell preparation from the peripheralblood of a donor, said mononuclear-enriched cell preparation comprisingat least 65% mononuclear cells;

freezing the mononuclear-enriched cell preparation in a freezing medium;

thawing the mononuclear-enriched cell preparation;

incubating the mononuclear-enriched cell preparation in an apoptosisinducing incubation medium comprising methylprednisolone at a finalconcentration of about 10-100 μg/mL;

wherein at least one of the freezing medium and the apoptosis inducingincubation medium comprise an anti-coagulant; and

suspending said cell preparation in an administration medium, therebyproviding the composition of the invention.

According to some embodiments, the apoptosis inducing incubation mediumused in the production method of the invention comprises ananti-coagulant. According to some embodiments, both the freezing mediumand apoptosis inducing incubation medium used in the production methodof the invention comprise an anti-coagulant. Without wishing to be boundby any theory or mechanism, in order to maintain a high and stable cellyield in different cell compositions, regardless of the cell collectionprotocol, it is preferable to add anti-coagulants to both the freezingmedium and apoptosis inducing incubation medium during production of thecomposition of the invention. According to some embodiments, a high andstable cell yield within the composition of the invention is a cellyield of at least 30%, preferably at least 40%, typically at least 50%cells of the initial population of cells used for induction ofapoptosis. Each possibility represents a separate embodiment of thepresent invention. As used herein, the terms “incubation medium” and“apoptosis inducing incubation medium” are used interchangeably.

According to some embodiments, the mononuclear-enriched cell compositionis frozen for at least about 6 hours. According to some embodiments, themononuclear-enriched cell composition is frozen for at least about 12hours. According to some embodiments, the mononuclear-enriched cellcomposition is frozen for about 12 hours. According to some embodiments,the mononuclear-enriched cell composition is frozen for at least 8, 10,12, 18, 24 hours. Each possibility represents a separate embodiment ofthe present invention.

According to some embodiments, incubating the thawed cells according tothe method of the invention is over a period of about 2-12 hours,possibly about 4-8 hours, typically for about 6 hours. Each possibilityrepresents a separate embodiment of the present invention. According tosome embodiments, incubating according to the method of the invention isincubating for about 6 hours. According to some embodiments, incubatingis for at least 6 hours.

According to some embodiments, obtaining a mononuclear-enriched cellpreparation is effected by leukapheresis. According to certainembodiments, obtaining a mononuclear-enriched cell preparation accordingto the production method of the invention refers to obtaining apreparation comprising at the time of collection at least 65%, possiblyat least 70%, typically at least 80% mononuclear cells. Each possibilityrepresents a separate embodiment of the present invention.

According to some embodiments, the freezing, according to the productionmethod of the invention, is the first step in inducing the earlyapoptotic state of the mononuclear cells in the cell preparation of theinvention. As used herein, the terms “freezing” and “cryopreservation”are used interchangeably.

According to some embodiments, the apoptosis inducing incubation mediumcomprises an apoptosis inducing agent, and incubation in such mediumpresents a second step in inducing the early apoptotic state of thecells in the cell preparation of the invention. According to someembodiments the apoptosis inducing agent is methylprednisolone.

According to some embodiments, the incubation medium comprisesmethylprednisolone at a final concentration of about 5-100 μg/mL,possibly about 40-60 μg/mL. Each possibility represents a separateembodiment of the present invention. According to some embodiments, theincubation medium comprises methylprednisolone at a final concentrationof about 50 μg/ml.

According to some embodiments, the cell concentration during theincubating is in the range of about 0.5×10⁶-10×10⁶. According to certainembodiments, the cell concentration during incubation is about 5×10⁶cells/ml.

According to another aspect, the present invention provides a method ofpreventing or ameliorating an immune disease or an autoimmune disease oran inflammatory disease in a subject in need thereof, comprisingadministering the pharmaceutical composition of the invention to thesubject. Each possibility represents a separate embodiment of thepresent invention.

According to some embodiments, the immune disease is GVHD. According tosome embodiments, the immune disease is high grade GVHD. According tosome embodiments, the present invention provides a method of preventingor ameliorating GVHD in a subject in need thereof, comprisingadministering the pharmaceutical composition of the invention to thesubject.

According to some embodiments, the GVHD is ameliorated to prevent theoccurrence of high grade GVHD. According to specific embodiments, highgrade GVHD is grade II-IV GVHD. According to another specificembodiment, high grade GVHD is grade III-IV GVHD. According to aparticular embodiment, the pharmaceutical composition induces a shiftfrom high grade GVHD to grade I GVHD. According to another embodiment,the GVHD is acute GVHD. According to yet another embodiment, the GVHD ischronic GVHD. According to another particular embodiment, the method ofadministering to the subject a pharmaceutical composition comprisingsaid mononuclear enriched cell preparation prevents high grade GVHDwhile the subject retains a graft-versus-tumor or graft-versus-leukemia(GVL) effect. According to some embodiments, following the treatmentmethod of the invention the subject retains the graft-versus-leukemia(GVL) effect.

According to another embodiment, the pharmaceutical composition reduceshepatotoxicity associated with GVHD. According to some embodiments, saidGVHD is liver GVHD.

According to another embodiment, the subject is undergoing hematopoieticstem-cell transplantation (HSCT). According to some embodiments, theHSCT is allogeneic HSCT. According to some embodiments, the HSCT isallogeneic HSCT and the pharmaceutical composition of the inventioncomprises cells obtained from the same donor of the hematopoieticstem-cells. According to particular embodiments, said subject issuffering from a hematopoietic malignancy. According to anotherembodiment, the hematopoietic malignancy is selected from leukemia,myelodysplastic syndrome (MDS), lymphoma, and multiple myeloma (i.e.,plasma cell dyscrasia). Each possibility represents a separateembodiment of the present invention. According to exemplary embodiments,said hematopoietic malignancy is selected from the group consisting ofacute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML),myelodysplastic syndrome (MDS) and chronic myelogenous leukemia (CML).Each possibility represents a separate embodiment of the presentinvention.

In other embodiments, the subject is undergoing solid organtransplantation. Solid organ transplantations include but are notlimited to an organ selected from lung, heart, kidney, pancreas, liverand small-bowel. Each possibility represents a separate embodiment ofthe present invention. According to some embodiments, the pharmaceuticalcomposition of the invention comprises cells obtained from the same or adifferent donor than the organ transplanted. According to someembodiments, the apoptotic cells of the invention are autologous cells.

According to another embodiment, the pharmaceutical composition isadministered following a conditioning treatment administered to thetransplantation recipient. According to another embodiment, thepharmaceutical composition is administered between one day prior totransplantation and 15 days following the transplantation. According toanother embodiment, the administering of the pharmaceutical compositionis carried out up to 30 hours prior to the transplantation. According tosome embodiments, the administering of the pharmaceutical composition iscarried out up to 24 hours prior to the transplantation. According to aparticular embodiment, the administering of the pharmaceuticalcomposition is carried out about 24-30 hours prior to thetransplantation. According to yet another embodiment, the administeringof the pharmaceutical composition is carried out at the same time as thetransplantation.

According to some embodiments the pharmaceutical composition isadministered intravenously. According to another embodiment, thepharmaceutical composition is administered in a single dose. Accordingto alternative embodiments the pharmaceutical composition isadministered in multiple doses. According to some embodiments, thepharmaceutical composition is formulated for intravenous injection.

According to additional embodiments, the inflammatory disease isarthritis, including, but not limited to, rheumatoid arthritis.According to some embodiments, the present invention provides a methodof preventing or ameliorating arthritis in a subject in need thereof,comprising administering the pharmaceutical composition of the inventionto the subject. According to another embodiment, the inflammatorydisease is gout. According to some embodiments, the present inventionprovides a method of preventing or ameliorating gout in a subject inneed thereof, comprising administering the pharmaceutical composition ofthe invention to the subject.

According to yet another embodiment, the inflammatory disease isinflammatory bowel disease. According to some embodiments, theinflammatory bowel disease is selected from Crohn's Disease, ulcerativecolitis and a combination thereof. Each possibility represents aseparate embodiment of the present invention. According to someembodiments, the present invention provides a method of preventing orameliorating an inflammatory disease selected from the group consistingof: Crohn's Disease and ulcerative colitis in a subject in need thereof,comprising administering the pharmaceutical composition of the inventionto the subject. Each possibility represents a separate embodiment of thepresent invention.

According to some embodiments, the present invention provides thepharmaceutical composition of the invention for use in preventing orameliorating an immune disease or an autoimmune disease or aninflammatory disease in a subject.

According to some embodiments, the present invention provides thepharmaceutical composition of the invention for use in preventing orameliorating GVHD in a subject. Each possibility represents a separateembodiment of the present invention. According to some embodiments, thepresent invention provides the pharmaceutical composition of theinvention for use in preventing or ameliorating an inflammatory boweldisease selected from the group consisting of: Crohn's Disease andulcerative colitis in a subject. Each possibility represents a separateembodiment of the present invention.

According to some embodiments, the present invention provides thepharmaceutical composition of the invention for the preparation of amedicament for preventing or ameliorating an immune disease or anautoimmune disease or an inflammatory disease. Each possibilityrepresents a separate embodiment of the present invention.

Other objects, features and advantages of the present invention willbecome clear from the following description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an in-vitro potency assay indicating inhibition ofimmature dendritic cell (iDC) maturation following interaction with theApoCell cell preparation. The tolerogenic effect of the apoptotic cellpreparation was examined following interaction with LPS treated iDCs,and detection of HLA-DR and CD86 expression levels. The results arerepresentative of one patient.

FIGS. 2A and 2B are histograms illustrating the increased rate ofoverall survival and non-relapsed survival, respectively, in patientsreceiving a single dose of apoptotic cells. Survival (percent) oftransplant patients in all four of cohorts I-IV (n=13), receiving35-210×10⁶ apoptotic cells prepared according to the methods disclosedherein is depicted at day 45, day 100 and day 180 of the study.

FIG. 2C is a histogram illustrating transplantation related mortality ofbone-marrow transplanted patients who received a single infusion of theApoCell composition (n=13; ApoCell) as compared to the survival rate ofmatched controls from hospital records (n=25; Historical Controls).

FIG. 3 is a histogram illustrating the time to first hospital dischargefor the treated patients in each cohort.

FIG. 4A is a histogram illustrating the reduced incidence of grade II-IVGVHD in transplant patients receiving a single high dose of apoptoticcells. The number (percent) of transplant patients developing acutegrade II-IV GVHD in each of four cohorts I (35×10⁶ apoptotic cells), II(70×10⁶ apoptotic cells), III (140×10⁶ apoptotic cells) and IV (210×10⁶apoptotic cells) receiving the apoptotic cell preparations. Notably, nopatients in the higher dosage treatment groups (cohorts III and IV)developed acute grade II-IV GVHD.

FIG. 4B is a histogram illustrating the reduced incidence of grade II-IVGVHD in 13 transplanted recipients receiving a single infusion ofapoptotic cell preparation (35-210×10⁶ apoptotic cells; ApoCell) ascompared to 25 matched historical controls from hospital records(Historical Controls).

FIG. 5A is a histogram illustrating the reduced incidence ofhepatotoxicity in transplant patients receiving a single dose ofapoptotic cells. The percentage of transplant patients developinghepatotoxicity in all four of cohorts I-IV (n=13; column 2), receivingfrom 35-210×10⁶ apoptotic cells preparations was compared with that ofmatched controls from hospital records (n=18; column 1), and with thelong-term documented transplant patients (n=1148; column 3) (Gooley etal, ibid.).

FIG. 5B is a histogram illustrating the reduced incidence ofhepatotoxicity in transplant patients receiving a single dose ofapoptotic cells. The incidence of hepatotoxicity in transplant patientsin each of four cohorts I (35×10⁶ apoptotic cells) (column 1), II(70×10⁶ apoptotic cells) (column 2), III (140×10⁶ apoptotic cells)(column 3) and IV (210×10⁶ apoptotic cells) (column 4) receivingapoptotic cells.

FIGS. 6A-H illustrate the kinetics of plasma biomarkers (TNFR1, IL-2Ra,IL-15, IL-1β, HGF, IL-8, IL-7, and IL-6) in post-transplant patientsreceiving a single dose of apoptotic cells.

FIGS. 7A-C are graphs illustrating the protective effect of theapoptotic cell preparation from T cell transfer-induced colitis. Rag1−/−mice received WT CD4⁺CD45RB^(low) (filled circles), or CD4⁺CD45RB^(high)T cells alone (filled squares) or in combination with apoptotic cells(filled triangles). (7A) Mean weight of indicated animal number pergroup (*p<0.05, t-test). (7B) IBD Clinical Score. Numbers inside boxesindicate the mean score of each parameter with error bar (*p<0.02,t-test). Data is presented as mean±SEM of 3 independent experiments.Weight change and stool consistency were monitored daily. Of note, nohematochezia was detected in either mice group. (7C) Illustratesincreased T regulatory cells in mesenteric lymph nodes following ApoCelltreatment compared to non-treated animals and control lymph nodes(popliteal).

FIG. 8 is a bar graph depicting dextran sodium sulfate (DSS) inducedcaspase-1-mediated IL-1β release from murine macrophages. Murine primaryresident peritoneal macrophages (pMΦ) were either left untreated, ortreated with the depicted combinations of: lipopolysaccharide (LPS), 3%DSS and z-YVAD-fmk (10 μM). IL-1β release was determined in thesupernatant by ELISA. Shown are representative data as means±SEM of 3independent experiments done in triplicate (*p<0.02, t test).

FIGS. 9A-D are bar graphs comparing IL-1β release by murine primaryresident peritoneal macrophages (pMΦ) treated with 3% DSS and/or LPS.The effect on IL-1β release was determined in the supernatant by ELISAfollowing treatment with either (9A) extracellular K⁺ (130 mM), (9B)bafilomycin A1 (10 nM) or (9C) ROS inhibitor N-acetyl-L-cysteine (NAC)(20 mM). (9D) IL-1β release following treatment with 3% DSS and/or LPSwas further determined in pMΦ extracted from wild type (WT) orNLRP3-deficient mice (Nlrp3−/−). Shown are representative data asmeans±SEM of 3 to 5 independent experiments done in triplicate (*p<0.05,**p<0.01, t test).

FIGS. 10A-D demonstrate the protective effect of the apoptotic cellpreparation from dextran sodium sulfate (DSS)-induced colitis. Balb/cmice were offered distilled water (filled circles), or distilled waterwith 3% DSS orally ad libitum with treatment of PBS (filled squares) orapoptotic cell (filled triangles). (10A) Mean weight of indicated animalnumber per group. (10B) IBD Clinical Score. Numbers inside boxesindicate the mean score of each parameter with error bar (*p<0.001,t-test). Data is presented as mean±SEM of 3 independent experiments.Weight change, hematochezia and stool consistency were monitored daily.(10C) Macroscopic changes of colon and spleen in DSS-treated mice.Photographs of the dissected large intestines and spleens of four micetreated with 3% DSS without- (DSS+PBS) or with apoptotic cell treatment(DSS+ApoCell). (10D) IL-1β cytokine level in colonic homogenate fromDSS-treated mice. Levels of IL-1β were analyzed by ELISA. Data ispresented as mean±SEM, 3 mice per group (*p<0.01, **p<0.001, one wayANOVA).

FIG. 11A shows the histological appearance and histological colitisseverity score of distal colon sections. H&E appearance (11A-I and11A-II) and histological score (11A-III) of distal colon sections areshown for DSS-treated Balb/c mice (11-AI, 3% DSS+PBS) and DSS- andApoCell-treated mice (11A-II, 3% DSS+ApoCell). Results from 3independent experiments (*p<0.05, unpaired t-test).

FIG. 11B demonstrates neutrophil accumulation inhibition in inflamedcolon treated by the apoptotic cell preparation. Mouse colon tissuesections were stained by immunohistochemistry assay using a rabbitmonoclonal antibody against mouse myeloperoxidase (MPO). Afterimmunostaining, slides were counterstained by hematoxylin. Images showthe MPO stain followed by HRP-anti rabbit secondary antibody. All imagesare ×200. (11B-I) Staining control. Untreated colon stained withHRP-anti rabbit secondary antibody only, without anti-MPO. (11B-II)Normal colon control. MPO-stained neutrophils in untreated colon (0%DSS+PBS). (11B-III) DSS treatment. MPO-stained neutrophils in 3% DSStreated colon (3% DSS+PBS). (11B-IV) Apoptotic cell & DSS treatment.MPO-stained neutrophils in 3% DSS-treated colon with apoptotic cellinfusion (3% DSS+ApoCell).

FIG. 12 shows cyclooxygenase-2 (COX-2) inhibition in DSS-induced colitistreated by the apoptotic cell preparation. Mouse colon tissue sectionswere stained by immunohistochemistry assay using an antibody againstmouse COX-2. After immunostaining, slides were counterstained byhematoxylin. All images are ×200. (I) Staining control. Untreated colonstained with HRP-anti rabbit secondary antibody only without anti COX-2.(II) COX-2 expression in untreated colon (0% DSS+PBS). (III) COX-2expression in 3% DSS treated colon (3% DSS+PBS). (IV) COX-2 expressionin 3% DSS-treated colon with apoptotic cell infusion (3% DSS+ApoCell).

FIG. 13 shows Iκ-Bα inhibition in DSS-induced colitis treated by theapoptotic cell preparation. Mouse colon tissue sections were stained byimmunohistochemistry assay using an antibody against mouse phospho-Iκ-Bα(pκ-Bα). After immunostaining, the slides were counterstained byhematoxylin. Images show pκ-Bα. All images are ×200. (I) Untreated colonstained with HRP anti-mouse secondary antibody only, withoutanti-pIκ-Bα. (II) pIκ-Bα staining in untreated colon (0% DSS+PBS). (III)pκ-Bα expression in 3% DSS-treated colon (3% DSS+PBS). (IV) pIκ-Bαexpression in 3% DSS treated colon with apoptotic cell infusion (3%DSS+ApoCell).

FIG. 14 shows NF-κB inhibition in DSS-induced colitis treated by theapoptotic cell preparation. Mouse colon tissue sections were stained byimmunohistochemistry assay using an antibody against mouse phospho-NF-κB(pNF-κB) p65. After immunostaining, slides were counterstained byhematoxylin. Images show pNF-κB p65 staining. All images are ×200. (I)Untreated colon stained with HRP-anti rabbit secondary antibody only,without anti NF-κB. (II) pNF-κB p65 staining in untreated colon (0%DSS+PBS). (III) pNF-κB p65 expression in 3% DSS-treated colon (3%DSS+PBS). (IV) pNF-κB p65 expression in 3% DSS treated colon withapoptotic cell infusion (3% DSS+ApoCell).

FIGS. 15A-E are bar graphs comparing the effect of apoptotic cells(ApoCell) on IL-1β release from murine pMΦ macrophages. (15A)Macrophages were treated with LPS and/or 3% DSS or treated withapoptotic cells (1:8) prior to LPS and/or 3% DSS treatment. Shown arerepresentative data as means±SEM of 3 independent experiments done intriplicate (*p<0.01, t-test). (15B) treated with LPS and/or 3% DSS ortreated with apoptotic cells (1:8) prior to LPS and/or 3% DSS treatment.In some treatments, cells were incubated with 2 μM cytochalasin D for 45min before the addition of apoptotic cells and DSS challenge. Shown arerepresentative data as means±SEM of 2 independent experiments done intriplicate (*p<0.01, one way ANOVA). (15C-E) pMΦ cells were incubatedeither in the presence of apoptotic cells for 2 h followed by LPSpriming for 1 h (black bar), or first primed with LPS for 1 h and thenincubated with apoptotic cells for 2 h (white bar). According to sometreatments, the pMΦ cells were then incubated with various inflammasomeinducers: Nigericin 2.5 μM (13C), monosodium urate (MSU) 200 μg/ml (15D)or calcium pyrophosphate dihydrate (CPPD) 200 μg/ml (15E). IL-1β wasdetermined in the supernatant by ELISA. Shown are representative data asmeans±SEM of 3 independent experiments done in duplicates (*p<0.001, oneway ANOVA.

FIGS. 16A-B depict western blot analyses performed on proteins extractedfrom supernatant and cell lysates of pMΦ cells. Some cells wereincubated either in the presence of apoptotic cells for 2 h followed byLPS priming for 1 h (sixth lane from left), or first primed with LPS (topromote NF-κB signaling) for 1 h and then incubated with apoptotic cellsfor 2 h (seventh lane from left). Following incubation with LPS and/orapoptotic cells, some of the pMΦ were incubated with (16A) nigericin 2.5μM or (16B) calcium pyrophosphate dihydrate 200 μg/mL (CPPD). Ananti-mouse actin served as a loading control. Shown are representativedata of two experiments.

FIGS. 17A-B depict western blot analyses performed on proteins extractedfrom supernatant and cell lysates of pMΦ cells. The pMΦ cells weretreated with either LPS, apoptotic cells or treated with apoptotic cellsprior to LPS (prior to NF-κB signaling, forth lane from left) or treatedwith LPS prior to apoptotic cell treatment (to promote NF-κB signaling,fifth lane from left). An anti-mouse actin served as a loading control.Shown are representative data of two experiments.

FIGS. 18A-F are micrographs depicting pMΦ cells as seen using in (18A)brightfield microscopy or (18B-F) under fluorescent microscopy stainedwith 1 μM of a reactive oxygen species (ROS) sensitive dye. (18B-F)Prior to staining with the ROS sensitive dye, the pMΦ cells were (18C)incubated with an ROS inhibitor (N-Acetyl-cysteine, 7.5 mM), (18D)treated with pyocyanin (0.5 mM), (18E) treated with 3% DSS or (18F)treated for 2 hours with apoptotic cells followed by a 30 min incubationwith 3% DSS. Magnification in all panels is ×100. The experiments wererepeated 3 times, independently; one representative experiment is shown.

FIG. 19 is a graph demonstrating reduction in reactive oxygen species(ROS) generation in DSS-treated macrophages pretreated with apoptoticcells. Flow-cytometry analysis of pMΦ stained with ROS-sensitive dye.pMΦ were treated for 2 h with apoptotic cells and primed with LPSfollowed by incubation with 3% DSS for 30 min. The negative controlsamples were treated with media only. ROS generation was determined byflow cytometry using a fluorescence probe, excluding dead cells base onFSC/SSC parameters. Shown are means±SEM of 3 experiments done intriplicates (*p<0.05, unpaired t-test).

FIGS. 20 A-B is bar graphs demonstrating the effect of the apoptoticcells on lysosomal damage and K+ efflux in peritoneal macrophages (pMΦ).(20A) Flow cytometry analysis of B6 pMΦ treated for 2 h with apoptoticcells and/or 24 h with DSS were stained with fluorochrome acridineorange (AO). Loss of fluorescence, which correlates with reduced numbersof lysosomes, was analyzed by flow cytometry, excluding dead cells baseon FSC/SSC parameters. Shown are means±SEM of 4 independent experiments(*p<0.05, **p<0.03 one way ANOVA). (20B) Apoptotic cell treatmentinhibits nigericin-induced IL-1β secretion. B6 pMΦ cells were treatedwith nigericin at the indicated concentrations in the presence of LPSpriming, with or without apoptotic cell treatment (*p<0.01 unpairedt-test).

DETAILED DESCRIPTION OF THE INVENTION

The present invention, in some embodiments thereof, relates to anapoptotic cell population and, more particularly, to early-apoptoticmononuclear enriched cell preparations. The present invention furtherrelates to methods for the production of said cell preparation, and theuse thereof in the clinical setting, in treatment of diseasescharacterized by pathological immune responses.

Transplantation-Related Diseases

Results of clinical trials of single-infusion of early-apoptoticmononuclear-enriched cells, produced by ex-vivo induction of apoptosisin donor cells, indicate the safety and efficacy of these apoptotic cellpopulations for prevention, prophylaxis and/or amelioration oftransplantation-related diseases, such as graft versus host disease(GVHD) in bone marrow transplant patients. As detailed herein below,induction of early-apoptosis in enriched mononuclear cells, according tothe methods of the present invention, provided a clinical gradepopulation of apoptotic allogeneic donor cells which, when infused withthe bone marrow derived cells from the same donor, affected importantfactors associated with transplantation, and effectively reduced theincidence of GVHD in subjects with hematological malignancies.

Particularly, at 100 days post transplantation, incidence of Grade II-IVGVHD was reduced in HSC transplant recipients treated with the apoptoticdonor cells prepared and the non-relapsed survival rate wassignificantly increased. As demonstrated herein, the incidences of acutegrades II through IV and grades III through IV GVHD were very low (23%and 15% respectively) in comparison with control (71% grade II-IV).Remarkably, treatment with higher dosages of the apoptotic cellpreparations (140×10⁶ and 210×10⁶ apoptotic cells) showed 0% acute GVHDgrade II-IV (compared to 50% of the matched historical controls).

Further, infusion of the apoptotic donor cells prepared according to themethods of the invention was effective in reducing the time toengraftment of the HSC and remarkably reducing the incidence ofhepatotoxicity in HSC transplant recipients.

While preventing induction of pro-inflammatory cytokines induced by GVHDhas been a challenge for clinical application, the results demonstratedherein show reduced serum levels of GVHD-related factors in HSCtransplant recipients who received the apoptotic cell compositions ofthe invention (FIG. 6). In particular, plasma levels of six differentbiomarkers: TNFRI, IL-2Ra, HGF, IL-8, IL-15 and IL-7, distinguished wellbetween high to low grade or no-GVHD. Additional two control cytokines(IL-1b and IL-6) further emphasized the specificity of findings.Further, in-vitro potency assay clearly showed inhibition of DCmaturation following interaction with the apoptotic cell preparations ofthe invention (FIG. 1).

According to some embodiments, the present invention provides a methodof preventing or ameliorating GVHD in a subject undergoing HSCT,comprising administering to the subject the pharmaceutical compositionof the invention.

According to some embodiments, the present invention provides a methodof preventing or ameliorating GVHD in a subject undergoing HSCT,comprising administering to the subject the pharmaceutical compositionof the invention. According to some embodiments, the present inventionprovides a method of preventing or ameliorating GVHD in a subjectundergoing HSCT, comprising administering to the subject thepharmaceutical composition of the invention comprising a cellpreparation comprising mononuclear enriched cells, wherein thepreparation comprises at least 85% mononuclear cells, wherein at least40% of the cells in the preparation are in an early apoptotic state,wherein at least 85% of the cells in the preparation are viable cellsand wherein the preparation comprises no more than 15% polymorphonuclearleukocytes. According to some embodiments, the present inventionprovides a method of preventing or ameliorating GVHD in a subjectundergoing HSCT, comprising administering to the subject thepharmaceutical composition of the invention comprising a cellpreparation comprising mononuclear enriched cells, wherein thepreparation comprises at least 85% mononuclear cells, wherein at least40% of the cells in the preparation are in an early apoptotic state andwherein at least 85% of the cells in the preparation are viable cells.

According to some embodiments, the present invention provides a methodof preventing or ameliorating GVHD in a subject undergoing HSCT,comprising administering to the subject a pharmaceutical compositioncomprising a cell preparation comprising mononuclear enriched cells,wherein the preparation comprises at least 85% mononuclear cells,wherein at least 40% of the cells in the preparation are in an earlyapoptotic state, wherein at least 85% of the cells in the preparationare viable cells and wherein the preparation comprises no more than 15%polymorphonuclear leukocytes; and wherein the pharmaceutical compositioncomprises an anti-coagulant selected from the group consisting of:heparin, ACD Formula A and a combination thereof. Each possibilityrepresents a separate embodiment of the present invention. According tosome embodiments, the present invention provides a method of preventingor ameliorating GVHD in a subject undergoing HSCT, comprisingadministering to the subject a pharmaceutical composition comprising acell preparation comprising mononuclear enriched cells, wherein thepreparation comprises at least 85% mononuclear cells, wherein at least40% of the cells in the preparation are in an early apoptotic state andwherein at least 85% of the cells in the preparation are viable cells;and wherein the pharmaceutical composition comprises an anti-coagulantselected from the group consisting of: heparin, ACD Formula A and acombination thereof. Each possibility represents a separate embodimentof the present invention.

According to some embodiments, the heparin in the pharmaceuticalcomposition of the invention is present at a concentration between 0.001U/ml and 3 U/ml, typically between 0.001 U/ml and 2.5 U/ml. According tosome embodiments, the heparin in the pharmaceutical composition of theinvention is present at a concentration between 0.005 U/ml and 2.5 U/ml.According to other embodiments, the heparin in the pharmaceuticalcomposition is present at a concentration between 0.01 U/ml and 1 U/ml.According to some embodiments, the ACD Formula A in the pharmaceuticalcomposition of the invention is present at a concentration of 0.01%-6%v/v. According to other embodiments, the ACD Formula A in thepharmaceutical composition is present at a concentration of 0.05%-5%v/v. According to other embodiments, the ACD Formula A in thepharmaceutical composition is present at a concentration of 0.01%-10%v/v.

According to some embodiments, the present invention provides a methodof preventing or ameliorating GVHD in a subject undergoing HSCT,comprising administering to the subject a pharmaceutical compositioncomprising a cell preparation comprising mononuclear enriched cells,wherein the preparation comprises at least 85% mononuclear cells,wherein at least 40% of the cells in the preparation are in an earlyapoptotic state, wherein at least 85% of the cells in the preparationare viable cells, wherein the preparation comprises no more than 15%polymorphonuclear leukocytes, and wherein the preparation comprisesmethylprednisolone at a concentration that does not exceed 30 μg/ml.According to some embodiments, the present invention provides a methodof preventing or ameliorating GVHD in a subject undergoing HSCT,comprising administering to the subject a pharmaceutical compositioncomprising a cell preparation comprising mononuclear enriched cells,wherein the preparation comprises at least 85% mononuclear cells,wherein at least 40% of the cells in the preparation are in an earlyapoptotic state, wherein at least 85% of the cells in the preparationare viable cells, and wherein the preparation comprisesmethylprednisolone at a concentration that does not exceed 30 μg/ml.

According to some embodiments, the present invention provides a methodof preventing or ameliorating GVHD in a subject undergoing HSCT,comprising administering to the subject a pharmaceutical compositioncomprising a cell preparation comprising mononuclear enriched cells,wherein the preparation comprises at least 85% mononuclear cells,wherein at least 40% of the cells in the preparation are in an earlyapoptotic state, wherein at least 85% of the cells in the preparationare viable cells and wherein the preparation comprises no more than 15%polymorphonuclear leukocytes, wherein the composition comprises ananti-coagulant selected from the group consisting of: heparin, ACDFormula A and a combination thereof, and wherein the compositioncomprises methylprednisolone at a concentration that does not exceed 30Each possibility represents a separate embodiment of the presentinvention. According to some embodiments, the present invention providesa method of preventing or ameliorating GVHD in a subject undergoingHSCT, comprising administering to the subject a pharmaceuticalcomposition comprising a cell preparation comprising mononuclearenriched cells, wherein the preparation comprises at least 85%mononuclear cells, wherein at least 40% of the cells in the preparationare in an early apoptotic state, wherein at least 85% of the cells inthe preparation are viable cells, wherein the composition comprises ananti-coagulant selected from the group consisting of: heparin, ACDFormula A and a combination thereof, and wherein the compositioncomprises methylprednisolone at a concentration that does not exceed 30μg/ml. Each possibility represents a separate embodiment of the presentinvention.

According to some embodiments, the present invention provides a methodof preventing or ameliorating GVHD in a subject undergoing HSCT,comprising administering to the subject a pharmaceutical compositioncomprising the cell preparation of the invention. According to someembodiments, the present invention provides a method of preventing orameliorating GVHD in a subject undergoing HSCT, comprising administeringto the subject the pharmaceutical composition of the invention.

According to some embodiments, the present invention provides thepharmaceutical composition of the invention for use in preventing orameliorating GVHD in a subject undergoing HSCT. According to someembodiments, the pharmaceutical composition of the invention furthercomprises residual methylprednisolone. According to some embodiments,the pharmaceutical composition of the invention further comprisesmethylprednisolone at a concentration that does not exceed 30 μg/ml.According to some embodiments, the pharmaceutical composition of theinvention further comprises an anti-coagulant. According to someembodiments, the anti-coagulant is selected from the group consistingof: heparin, ACD Formula A and a combination thereof. Each possibilityrepresents a separate embodiment of the present invention.

According to some embodiments, the GVHD is high grade GVHD. According tospecific embodiments, high grade GVHD is grade II-IV GVHD. According toanother specific embodiment, high grade GVHD is grade III-IV GVHD.According to a particular embodiment, the pharmaceutical composition ofthe invention induces a shift from high grade GVHD to grade I GVHD.

According to another embodiment, the GVHD is acute GVHD. According toyet another embodiment, the GVHD is chronic GVHD. According to anotherparticular embodiment, a subject administered with the pharmaceuticalcomposition of the invention retains a graft-versus-tumor (GVTS) orgraft-versus-leukemia (GVL) effect. Each possibility represents aseparate embodiment of the present invention.

According to some embodiments, the GVHD is GVHD in the liver of thesubject. Liver dysfunction in allogeneic HSCT recipients may be due to avariety of factors including toxicity from the preparative regimen andother medications, infection, veno-occlusive disease (VOD), and acuteand chronic graft-versus-host disease (GVHD) of the liver.

According to another embodiment, the pharmaceutical composition of theinvention reduces hepatotoxicity associated with GVHD. According to someembodiments, the cell preparation of the invention reduceshepatotoxicity associated with GVHD. Common symptoms and complicationsof Hepatotoxicity include lymphadenitis, fever, red blood cellsedimentation rate increased high bilirubin levels and febrileneutropenia.

According to some embodiments, the present invention provides a methodof preventing or ameliorating an immune disease or an autoimmune diseaseor an inflammatory disease in a subject in need thereof, comprisingadministering to the subject a pharmaceutical composition comprising thecell preparation of the invention. Each possibility represents aseparate embodiment of the present invention. According to someembodiments, the present invention provides a method of preventing orameliorating an immune disease or an autoimmune disease or aninflammatory disease in a subject in need thereof, comprisingadministering to the subject the pharmaceutical composition of theinvention. Each possibility represents a separate embodiment of thepresent invention.

According to some embodiments, the present invention provides the cellpreparation of the invention for use in preventing or ameliorating animmune disease or an autoimmune disease or an inflammatory disease in asubject in need thereof. Each possibility represents a separateembodiment of the present invention. According to some embodiments, thepresent invention provides the pharmaceutical composition of theinvention for use in preventing or ameliorating an immune disease or anautoimmune disease or an inflammatory disease in a subject in needthereof. Each possibility represents a separate embodiment of thepresent invention.

According to some embodiments, the immune disease is GVHD. According tosome embodiments, the present invention provides the pharmaceuticalcomposition of the invention for use in preventing or ameliorating GVHDin a subject in need thereof.

According to some embodiments, the present invention provides a methodof preventing or ameliorating a hematopoietic malignancy comprisingadministering to a subject in need thereof the pharmaceuticalcomposition of the invention. Each possibility represents a separateembodiment of the present invention. According to particularembodiments, the subject is suffering from a hematopoietic malignancy.

The term “hematopoietic malignancy” as used herein refers to any bloodcell cancer, characterized by uncontrolled, abnormal growth of bloodcells. The term “hematopoietic malignancy” includes but is not limitedto leukemia, myelodysplastic syndrome, lymphoma, and multiple myeloma(plasma cell dyscrasia). The term “leukemia” refers to a disease of theblood forming organs characterized by an abnormal increase in the numberof leucocytes in the tissues of the body with or without a correspondingincrease of those in the circulating blood (e.g., acute lymphoblasticleukemia, ALL; acute myelogenous leukemia, AML; chronic myelogenousleukemia, CML; etc.). The term “myelodysplastic syndrome” refers to acondition in which the bone marrow shows qualitative and quantitativechanges suggestive of a preleukemic process, but having a chronic coursethat does not necessarily terminate as acute leukemia. The term“lymphoma” refers to a malignant tumor of lymphoblasts derived from B orT lymphocytes (e.g., Hodgkin lymphoma, HL; non-Hodgkin lymphoma, NHL;etc.). The term “plasma cell dyscrasia” refers to plasmacytosis due toplasma cell proliferation (e.g., multiple myeloma, MM; plasma cellleukemia, PCL; etc.)

According to exemplary embodiments, said hematopoietic malignancy isselected from the group consisting of MDS, acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML) and chronic myelogenous leukemia(CML).

Infusion of certain types of the donor blood cells such as T-lymphocytescan also stimulate a graft-versus-leukemia effect. This effect has beenbest observed in patients with chronic myeloid leukemia (CML). In CML,75 percent of patients relapsing after transplant re-enter remission.For other disorders such as acute myeloid leukemia (AML) andmyelodysplastic syndrome (MDS), the effect is less pronounced; AML andMDS in approximately 20 percent of patients enter remission. Forpatients with acute lymphoblastic leukemia (ALL), the presence ofgraft-vs-leukemia effect is unclear, although small numbers of patientshave reportedly benefited, at least transiently, from the effect.

In other ways, the donor immune cells may recognize residual leukemia,lymphoma or cancer cells as being different and destroy them.Retrospective studies have demonstrated that patients in whom acute orchronic GVHD develops have lower disease recurrence rates than patientswho do not develop GVHD. This finding is an indirect indication of agraft-versus-tumor effect.

The term “conditioning treatment” refers to preparative treatment oftransplant recipient with various conditioning regimens includingradiation, immune sera, chemotherapy, and/or immunosuppressive agents,prior to transplantation. Transplantation conditioning is very commonbefore bone marrow transplantation.

As used herein, the terms “subject”, “patient” and “subject in needthereof” may be used interchangeably and refer to a subject in need ofadministration of the pharmaceutical composition of the invention.

According to some embodiments, the pharmaceutical composition of theinvention is administered to a subject who has undergone or will undergoHSCT. Each possibility represents a separate embodiment of the presentinvention. According to some embodiments, a subject in need thereof is asubject undergoing HSCT. According to some embodiments, theHematopoietic Stem Cells (HSCs) transplanted into a subject in needthereof and the cells of the pharmaceutical composition of the inventionare derived from the same donor.

According to another embodiment, administering of the pharmaceuticalcomposition of the invention is carried out up to 24 hours prior to theHSCT. According to some embodiments, administering of the pharmaceuticalcomposition of the invention is carried out about 24-30 hours prior tothe HSCT. According to yet another embodiment, the administering of thepharmaceutical composition of the invention is carried out at the sametime as the HSCT. According to some embodiments, the administering ofthe pharmaceutical composition of the invention is carried out up to 15days following the HSCT. According to additional embodiments, the HSCsused in the HSCT are allogeneic HSCs. According to non-limitingexamples, the HSCs used in the HSCT may be derived from bone marrow,peripheral blood, or umbilical cord blood. Each possibility represents aseparate embodiment of the present invention. According to anotherembodiment, the pharmaceutical composition of the invention isadministered in a single dose.

Inflammatory Bowel Diseases

Inflammatory bowel diseases (IBD) are characterized by chronicintestinal inflammation with dysregulation of the mucosal immune systemin the gastrointestinal tract manifested as Crohn's disease andulcerative colitis. As used herein, the term IBD refers to Crohn'sdisease, ulcerative colitis or a combination thereof. Each possibilityrepresents a separate embodiment of the present invention. Geneticfactors and environmental factors that include both intestinalmicroflora and danger signals such as dextran sodium sulfate (DSS) wereall shown to induce intestinal inflammation. TNFα and IFNγ blockade andanti-IL-1β strategies, as well as antibiotic treatment were able toameliorate colitis induction, suggesting a role for nuclear factor-kappaB (NF-κB) and inflammasome inhibition of macrophages and dendritic cellsin the lamina propria.

The therapeutic effect of the cell preparation and pharmaceuticalcomposition of the invention disclosed herein was further demonstratedin inflammatory bowel disease (IBD). As exemplified herein below, asingle infusion of the pharmaceutical composition of the inventionsignificantly ameliorated both the clinical score and histologicalappearance of two different models of IBD: adoptive T cell transfer(TCT) of naïve CD4 cells and dextran sulfate sodium (DSS)-inducedcolitis.

The dextran sulfate sodium (DSS) model is generally viewed as anepithelial damage model suited to investigate wound-healing processesand innate immune responses. It has been suggested that DSS uptake bylamina propria macrophages initiates an inflammatory process.Macrophages primed with LPS and subsequently exposed to DSS secrete highlevels of IL-1β and IL-18 in an NLRP3-, ASC-, and caspase-1-dependentmanner. This effect was completely abrogated when the endocytosis of DSSwas experimentally blocked.

Caspases are cysteine proteases first shown to be involved in theinduction and execution of programmed cell death, and later ininflammation, leading to their categorization as proapoptotic orproinflammatory. Caspase-1 is one of three proinflammatory caspases thathave been well studied and characterized. Its catalytic activity isregulated by autoactivation within multiprotein complexes called“inflammasomes” that mediate caspase-1-dependent processing ofcytokines, most notably IL-1β. Proinflammatory stimuli induce expressionof the IL-1β preform, but cytokine maturation and release are controlledby the inflammasome.

A number of node-like receptors (NLR) family members have been reported,but their physiological functions in vivo have been elucidated in only afew cases. NLRP1, NLRP3, and IPAF are danger sentinels thatself-oligomerize via homotypic NACHT domain interactions to form highmolecular weight complexes that induce caspase-1 autoactivation. NLRP3inflammasome consists of the NLRP3 scaffold, the ASC (PYCARD) adaptor,and caspase-1. As part of immune defense, NLRP3 is activated uponexposure to whole pathogens such as the fungi Candida albicans andSaccharomyces cerevisiae, pore-forming toxins, and viruses, as well asdiverse microbial components. Interestingly, NLRP3 is also activated byhost-derived molecules leading to inflammatory disorders and perhapsautoimmune diseases. Extracellular ATP and hyaloronan, released byinjured cells, monosodium urate crystals, and fibrillar amyloid-bpeptides, are some examples.

The present invention demonstrates, for the first time, that theapoptotic cell composition of the invention negatively regulates theNLRP3 inflammasome, both in vitro and in vivo, and is able todownregulate the pro-inflammatory response induced via NLRP3inflammasome in hematopoietic cells.

The inflammasome triggering is a two-hit model requiring bothToll-Like-Receptor (TLR) and inflammasome triggering. Indeed, theapoptotic cells of the invention were shown to inhibit TLRs and theNF-κB pathway. TLR triggering is important for enhanced transcription ofpro-IL-1β and pro-IL-18, and is in fact needed for the DSS effect. It isnow demonstrated herein that the apoptotic cell preparation of theinvention inhibited the secretion of activated IL-1β at both pre- andpost-transcription levels and had distinct inhibition effects on NF-κBand NLRP3.

It is further demonstrated herein below, that the apoptotic cellcomposition of the invention affects all three molecular mechanismsdescribed in the regulation of inflammasome activation. Apoptotic cells,as comprised in the composition of the invention, were shown to becapable of reducing and inhibiting the formation of ROS, at ratessimilar to those shown for the chemical inhibitor NAC. It is wellestablished that macrophages make use of toxic ROS to control microbialpathogens as part of the innate immune response and ROS were identifiedas major mediators of inflammatory signals believed to play a role inthe development of IBD. Furthermore, generation of ROS was found toinduce IL-1β via ERK phosphorylation. On the other hand, IL-1β signalsmay induce ROS generation. While it has been shown that DSS inducesformation of ROS, a marked reduction in ROS generation, and consequentlyless IL-1β secretion, was observed when macrophages were pretreated withapoptotic cells as comprised in the preparation of the invention.

The second mechanism involves the lysosome. It was shown that lysosomaldamage or leakage may serve as an endogenous danger signal and is sensedby the NLRP3 inflammasome. The involvement of the lysosome vacuole wasanalyzed since apoptotic cell clearances are mediated via thephagolysosomal pathway. As exemplified herein, lysosomes from peritonealmacrophages that had engulfed apoptotic cells were more stable to DSSchallenge, and were not affected or damaged. This may point to thenotion that engulfed apoptotic cells desensitize lysosome for at least24 h after apoptotic ingestion. Taken together, these resultsdemonstrate a mechanism of inflammasome inhibition and resolution ofinflammation stemming from apoptotic cell clearance.

Inflammasomes were also suggested to be activated in response tosignaling pathways that deplete intracellular potassium, such as thepotassium ionophore nigericin. When macrophages were pretreated withapoptotic cells, nigericin-induced IL-1β secretion was significantlyinhibited. The means by which apoptotic cells inhibit nigericin-inducedIL-1β secretion in not clear, but supporting evidence might illustrate adirect inflammasome upstream inhibitory mechanism that is perhaps bestmediated via NF-κB signaling. This observation illustrates a mechanismof regulation of inflammation that could take place in both infectiousand non-infectious inflammatory conditions. Failure to clear apoptoticcells will trigger persistence inflammasome-dependent inflammation asperhaps is seen in failure to clear intracellular organelles.

In summary, infusion of the cell preparation of the invention isbeneficial in mice models of IBD and inhibits both inflammasome- andNF-κB-dependent inflammation via three mechanisms.

According to some embodiments, the present invention provides a methodof preventing or ameliorating IBD in a subject in need thereof,comprising administering to the subject the pharmaceutical compositionof the invention. According to some embodiments, the present inventionprovides the pharmaceutical composition of the invention for use inpreventing or ameliorating IBD in a subject in need thereof. Eachpossibility represents a separate embodiment of the present invention.

According to some embodiments, the present invention provides a methodof preventing or ameliorating IBD in a subject in need thereof,comprising administering to the subject a pharmaceutical compositioncomprising a cell preparation comprising mononuclear enriched cells,wherein the preparation comprises at least 85% mononuclear cells,wherein at least 40% of the cells in the preparation are in an earlyapoptotic state, wherein at least 85% of the cells in the preparationare viable cells and wherein the preparation comprises no more than 15%polymorphonuclear leukocytes; and wherein the pharmaceutical compositioncomprises an anti-coagulant selected from the group consisting of:heparin, ACD Formula A and a combination thereof. Each possibilityrepresents a separate embodiment of the present invention.

According to some embodiments, the present invention provides a methodof preventing or ameliorating IBD in a subject in need thereof,comprising administering to the subject a pharmaceutical compositioncomprising a cell preparation comprising mononuclear enriched cells,wherein the preparation comprises at least 85% mononuclear cells,wherein at least 40% of the cells in the preparation are in an earlyapoptotic state, wherein at least 85% of the cells in the preparationare viable cells and wherein the preparation comprises no more than 15%polymorphonuclear leukocytes, wherein the composition comprises ananti-coagulant selected from the group consisting of: heparin, ACDFormula A and a combination thereof, and wherein the preparationcomprises methylprednisolone at a concentration that does not exceed 30Each possibility represents a separate embodiment of the presentinvention.

Apoptotic Cell Preparations

According to some embodiments, the preparation of the invention refersto a cell preparation comprising mononuclear enriched cells, wherein thepreparation comprises at least 85% mononuclear cells, wherein at least40% of the cells in the preparation are in an early apoptotic state,wherein at least 85% of the cells in the preparation are viable cellsand wherein the preparation comprises no more than 15% polymorphonuclearleukocytes. According to certain embodiments, the preparation of theinvention refers to a cell preparation comprising mononuclear enrichedcells, wherein the preparation comprises at least 85% mononuclear cells,wherein at least 40% of the cells in the preparation are in an earlyapoptotic state, wherein at least 85% of the cells in the preparationare viable cells and wherein the preparation comprises no more than 15%CD15^(high) expressing cells. As used herein, the terms “thepreparation”, “the preparation of the invention”, “the apoptotic cellpreparation of the invention”, “the cell preparation of the invention”,“the cell preparation” and “the mononuclear enriched preparation” areused interchangeably.

According to some embodiments, the present invention provides a cellpreparation comprising mononuclear enriched cells, wherein thepreparation comprises at least 85% mononuclear cells, wherein at least40% of the cells in the preparation are in an early apoptotic state,wherein at least 85% of the cells in the preparation are viable cellsand wherein the preparation comprises no more than 15% polymorphonuclearleukocytes.

As used herein, the terms “the composition of the invention”, “thepharmaceutical composition of the invention”, “the pharmaceuticalcomposition”, “the composition”, “apoptotic cell composition” and“composition comprising the cell preparation of the invention” are usedinterchangeably and refer to a composition comprising the cellpreparation of the invention. According to some embodiments, thepharmaceutical composition of the invention refers to a compositioncomprising the cell preparation of the invention and further comprisingan anticoagulant. According to some embodiments, the term “thecomposition of the invention” refers to a composition comprising thecell preparation of the invention and a final suspension medium used foradministration of the cell preparation to a patient. According to someembodiments, the terms “final suspension medium” and “administrationmedium”, as used herein, are used interchangeably and refer to themedium used for administration of the cell preparation of the inventionto a subject. According to some embodiments, a pharmaceuticalcomposition comprising the cell-preparation of the invention is referredto herein as “ApoCell”. According to some embodiments, thepharmaceutical composition of the invention is referred to herein as“ApoCell”.

In some embodiments, the mononuclear enriched cell preparation of theinvention comprises at least 85% mononuclear cells, at least 85%mononuclear cells, alternatively at least 90% mononuclear cells or atleast 95% mononuclear cells, wherein each possibility is a separateembodiment of the invention. According to some embodiments, themononuclear enriched cell preparation of the invention comprises atleast 80% mononuclear cells. According to some embodiments, themononuclear enriched cell preparation of the invention comprises atleast 90% mononuclear cells. According to some embodiments, themononuclear enriched cell preparation of the invention comprises atleast 95% mononuclear cells. According to some embodiments, the cellpreparation of the invention comprises at least one cell type selectedfrom the group consisting of: lymphocytes, monocytes and natural killercells. According to some embodiments, mononuclear cells compriselymphocytes and monocytes. As used herein and in the claims, mononuclearcells are leukocytes having a one lobed nucleus.

According to some embodiments, at least 40% of the cell preparation ofthe invention are in an early apoptotic state. According to otherembodiments, at least 40%, preferably around 50% of the cell preparationof the invention are in an early apoptotic state. Each possibilityrepresents a separate embodiment of the present invention. According tosome embodiments, between 40-65%, preferably between 55-60% of the cellpreparation of the invention are in an early apoptotic state. Eachpossibility represents a separate embodiment of the present invention.

It should be appreciated that, according to some embodiments, the highpercentage of mononuclear cells in the cell preparation disclosed hereinis achieved following the multistep manufacturing protocol, as describedherein below (including leukapheresis, early-apoptosis induction usingcryopreservation and incubation with methylprednisolone and variouswashing steps).

According to some embodiments, the mononuclear-enriched cell preparationof the invention comprises low concentrations of non-mononuclearleukocytes such as, but not limited to, polymorphonuclear leukocytes andneutrophils. Preferably, said mononuclear enriched cell preparation isdevoid of granulocytes.

According to some embodiments, granulocytes disintegrate during varioussteps of the production method of the invention. According to someembodiments, the composition of the invention comprises no more than15%, alternatively no more than 10%, typically no more than 5%granulocytes. Each possibility represents a separate embodiment of thepresent invention. According to some embodiments, granulocytesdisintegrate to a significant degree following the freezing and thawingsteps of the production method of the invention. According to someembodiments, granulocytes disintegrate to a significant degree followingthe freezing and thawing steps of the production method of theinvention, and are washed from the preparation of the invention duringwash steps after the freezing and/or thawing steps. Each possibilityrepresents a separate embodiment of the present invention. According tosome embodiments, disintegrated granulocytes are washed from the cellpreparation of the invention during various washing steps of theproduction method of the invention.

According to some embodiments, the composition of the inventioncomprises no more than 15%, possibly no more than 10%, typically no morethan 5% polymorphonuclear leukocytes. Each possibility represents aseparate embodiment of the present invention. According to someembodiments, the composition of the invention comprises no more than 5%polymorphonuclear leukocytes.

According to some embodiments, the composition of the inventioncomprises no more than 15%, alternatively no more than 10%, typically nomore than 5% CD15^(high) expressing cells. Each possibility represents aseparate embodiment of the present invention. According to someembodiments, the composition of the invention comprises no more than 5%CD15^(high) expressing cells. According to some embodiments, asexemplified herein below, the composition of the invention comprises nomore than 1% CD15^(high) expressing cells. As used herein and in theclaims, CD15^(high) expressing cells are granulocytes.

An early feature of apoptosis is a morphological change in the plasmamembrane. This change involves the translocation of the membranephospholipid phosphatidylserine (PS) from the internal layer to theexternal layer of the cell membrane. In the presence of calcium ions,Annexin V has a high specificity and affinity for PS. Thus, the bindingof Annexin V to cells with exposed PS provides a very sensitive methodfor detecting early cellular apoptosis. Thus, an “early apoptotic state”of a cell or “early apoptotic cells”, as used herein, refers to a cellpopulation which still have intact cell membranes, but have started toundergo DNA cleavage and have started to undergo translocation ofphosphatidylserine. As used herein, early apoptotic cells, or cells atan early apoptotic state, are cells which are stained positively usingAnnexin V and are stained negatively with propidium iodide (PI). Methodsfor detection of early apoptosis are known in the art, such as earlyapoptotic cell detection of annexin V positive and propidium iodide (PI)negative, by flow cytometry. According to some embodiments, cells whichare in a late apoptotic state may be detected by a positive stainingusing annexin V and a positive staining using PI as may be evidencedusing flow cytometry. It is to be noted that PI is membrane impermeableand thus is only able to enter cells in which the intactness of the cellmembrane has been compromised, such as in late apoptotic or necroticcells. According to some embodiments, necrotic cells show strongstaining for PI, as may be evidenced using flow cytometry.

According to some embodiments, at least 40% of the cells in the cellpreparation of the invention are in an early-apoptotic state. Accordingto other embodiments, at least 50% or alternatively at least 60% of thecells in the cell preparation of the invention are in an early-apoptoticstate. Each possibility represents a separate embodiment of the presentinvention. In some embodiments, the cell preparation of the inventioncomprises cells in suspension. In another embodiment, the cellpreparation of the invention is not prepared by inducing cells to adhereto a surface.

As used herein, “viability” of the cells refers to cells not undergoingnecrosis or late apoptosis. Accordingly, the term “viable cells”, asused herein, refers to cells not undergoing necrosis or cells which arenot in a late apoptotic state. According to some embodiments, the term“viable cells” refers to cells having an intact plasma membrane. Assaysfor determining cell viability are known in the art, such as usingpropidium iodide (PI) staining which may be detected by flow cytometry.Accordingly, according to some embodiments, viable cells are cells whichdo not show propidium iodide intake. Necrosis can be further identified,by using light, fluorescence or electron microscopy techniques, or viauptake of the dye trypan blue.

Apoptosis, which is a distinct cell death process from necrosis, is theprogrammed and orderly physiological elimination of cells, occurring,for example, during normal cell and tissue development, T-lymphocytekilling of pathogen-infected cells, and self-elimination of mutationallydamaged cells. Apoptotic cells are characterized by distinct morphologicalterations in the cytoplasm and nucleus, chromatin cleavage atregularly spaced sites, and endonucleolytic cleavage of genomic DNA atinternucleosomal sites. Assays for determining cell apoptosis are knownin the art, such as using AnnexinV. Necrosis, on the other hand, is aninherently pathological and pro-inflammatory process of cell deathcaused, typically but not exclusively, by the uncontrolled, progressivedegradative action of enzymes following lethal cellular injury. Necroticcells are typically characterized by mitochondrial swelling, nuclearflocculation, cell lysis, loss of membrane integrity, and ultimatelycell death.

According to some embodiments, the cell preparation of the inventioncomprises at least 85%, 90% 95% viable cells, alternatively at least 97%viable cells. Each possibility represents a separate embodiment of thepresent invention. In another embodiments, the cell preparation of theinvention comprises at-most 15%, 10%, 5% necrotic cells or cells in alate apoptotic state, alternatively at most 3% necrotic cells or cellsin a late apoptotic state. Each possibility represents a separateembodiment of the present invention. In additional embodiments, the highpercentage of viable cells in the cell preparation of the inventionremains for at least 24 hours following preparation. According to someembodiments, necrotic cells and/or cells in a late apoptotic statedisintegrate and are thus substantially eliminated from the finalcell-preparation of the invention during washing steps of the productionmethod of the invention. Each possibility represents a separateembodiment of the present invention.

According to some embodiments of the present invention, in order toinduce therapeutic immune tolerance in autoimmune diseases, such asGVHD, the therapeutic mononuclear enriched cells in the cell preparationof the invention are preferably derived from an allogeneic individual.Allogeneic mononuclear enriched cells are preferably haplotype-matchedwith the subject receiving said cells. Haplotype-matching of humansubjects is routinely practiced in the art in the context of therapeutictransplantation, and usually involves matching of HLA-A, HLA-B, andHLA-DR alleles. In some embodiments, the source of the mononuclearenriched cell preparation is derived from an allogeneic donor that isHLA-matched at least a 7/8 at the HLA A, B, C, and DR loci. In someembodiments, the source of the mononuclear enriched cell preparation isautologous.

According to some embodiments, the pharmaceutical composition of theinvention comprises the cell preparation of the invention and furthercomprises an anti-coagulant. According to some embodiments, thepharmaceutical composition of the invention comprises the cellpreparation of the invention and further comprises residualmethylprednisolone. According to other embodiments, the pharmaceuticalcomposition of the invention comprises the cell preparation of theinvention and further comprises an anti-coagulant and residualmethylprednisolone. According to some embodiments, residualmethylprednisolone refers to methylprednisolone remaining in thecomposition of the invention following use of the production method ofthe invention.

According to some embodiments, the composition of the inventioncomprises an anti-coagulant. As known in the art, an anti-coagulant, asused herein, refers to a substance which prevents or decreases bloodclotting. According to some embodiments, the anti-coagulant is heparin.According to other embodiments, the anti-coagulant isAcid-Citrate-Dextrose (ACD), formula A. According to some embodiments,the anti-coagulant is a composition comprising ACD formula A andheparin. According to some embodiments, the anti-coagulant is ACDformula A containing heparin at a concentration of about 10 U/ml.According to some embodiments, the anti-coagulant is selected from thegroup consisting of: heparin, ACD Formula A and a combination thereof.Each possibility represents a separate embodiment of the presentinvention. According to some embodiments, the presence of ananti-coagulant in the composition of the invention is due to addition ofthe anti-coagulant during the freezing and/or incubation and/or washingstages of the composition's production process. Each possibilityrepresents a separate embodiment of the present invention. According tosome embodiments, the presence of an anti-coagulant during production ofthe composition of the invention does not adversely affect apoptosisinduction as described herein.

According to some embodiments, the composition of the inventioncomprises heparin. According to some embodiments, heparin is selectedfrom the group consisting of: sulfated heteropolysaccharide heparin,unfractionated heparin (UFH), low molecular weight heparin (LMWH) and acombination thereof. Each possibility represents a separate embodimentof the present invention. According to other embodiments, heparin is asynthetic heparin, such as, but not limited to, Fondaparinaux.

According to some embodiments, the composition of the inventioncomprises heparin at a concentration between 0.001 U/ml and 3 U/ml,alternatively between 0.005 U/ml and 2.5 U/ml, typically between 0.01U/ml and 1 U/ml. Each possibility represents a separate embodiment ofthe present invention. According to other embodiments, the compositionof the invention comprises heparin at a concentration between 0.001-2.5U/ml, alternatively between 0.001-1 U/ml, possibly between 0.001-0.5U/ml. Each possibility represents a separate embodiment of the presentinvention.

According to other embodiments, the composition of the inventioncomprises heparin at a concentration between 0.005-1 U/ml, alternativelybetween 0.005-0.6 U/ml, possibly between 0.005-0.5 U/ml. Eachpossibility represents a separate embodiment of the present invention.According to other embodiments, the composition of the inventioncomprises heparin at a concentration between 0.01-3 U/ml, alternativelybetween 0.01-2 U/ml or between 0.01-0.6 U/ml. Each possibilityrepresents a separate embodiment of the present invention. According tosome embodiments, the composition of the invention comprises heparin ata concentration between 0.01-0.5 U/ml. According to some embodiments,the composition of the invention comprises heparin at a concentrationbetween 0.05 U/ml and 0.25 U/ml. According to certain embodiments, thecomposition of the invention comprises heparin at a concentrationbetween 0.01 U/ml and 0.6 U/ml.

According to some embodiments, the composition of the inventioncomprises up to 3 U/ml heparin, typically up to 2.5 U/ml heparin,possibly up to 1 U/ml heparin, alternatively up to 0.5 U/ml heparin.According to some embodiments, the composition of the inventioncomprises at least 0.001 U/ml heparin, alternatively at least 0.005 U/mlheparin, possibly at least 0.01 heparin. Each possibility represents aseparate embodiment of the present invention. According to someembodiments, the composition of the invention comprises up to 300 U,alternatively up to 150 U, possibly up to 75 U of Heparin. Eachpossibility represents a separate embodiment of the present invention.According to certain embodiments, the composition of the inventioncomprises up to 180 U of heparin.

According to some embodiments, heparin comprised in the composition ofthe invention refers to heparin in the composition comprising the cellpreparation of the invention and the final suspension medium used foradministration of the cell preparation to a patient. According to someembodiments, ACD Formula A comprised in the composition of the inventionrefers to heparin in the composition comprising the cell preparation ofthe invention and the final suspension medium used for administration ofthe cell preparation to a patient.

According to some embodiments, the composition of the inventioncomprises between 0.5-500 U of heparin, possibly between 0.5-500 U ofheparin, alternatively between 7-180 U of heparin. Each possibilityrepresents a separate embodiment of the present invention.

According to some embodiments, the composition of the inventioncomprises ACD Formula A. According to some embodiments, ACD Formula Acomprises citric acid, dextrose and sodium citrate. According to someembodiments, ACD Formula A comprises anhydrous citric acid at aconcentration of 0.73 gr/100 ml, dextrose monohydrate at a concentrationof 2.45 gr/100 ml and sodium citrate dehydrate at a concentration of2.20 gr/100 ml.

According to some embodiments, the composition of the inventioncomprises ACD formula A at a concentration between 0.01%-10% v/v,alternatively between 0.05%-6% v/v, possibly between 0.1%-5% v/v. Eachpossibility represents a separate embodiment of the present invention.According to other embodiments, the composition of the inventioncomprises ACD formula A at a concentration between 0.05%-10% v/v,possibly 0.05%-6% v/v, alternatively between 0.05%-5% v/v. Eachpossibility represents a separate embodiment of the present invention.

According to alternate embodiments, the composition of the inventioncomprises ACD formula A at a concentration between 0.1%-10% v/v,alternatively between 0.1%-6%, possibly between 0.1%-5% v/v. Eachpossibility represents a separate embodiment of the present invention.According to some embodiments, the composition of the inventioncomprises ACD formula A at a concentration between 0.5%-2.5% v/v.According to certain embodiments, the composition of the inventioncomprises ACD formula A at a concentration between 0.05%-6% v/v,typically between 0.1%-6% v/v. Each possibility represents a separateembodiment of the present invention.

According to some embodiments, the composition of the inventioncomprises up to 15 ml, alternatively up to 9 ml, possibly up to 7.5 mlof ACD formula A. Each possibility represents a separate embodiment ofthe present invention. According to certain embodiments, the compositionof the invention comprises up to 18 ml of ACD formula A.

According to some embodiments, the composition of the inventioncomprises between 0.05-40 ml of ACD formula A, possibly between 0.1-25ml of ACD formula A, alternatively between 0.7-18 ml of ACD formula A.Each possibility represents a separate embodiment of the presentinvention.

According to some embodiments, the composition of the invention furthercomprises methylprednisolone. According to some embodiments, thepresence of residual methylprednisolone in the composition of theinvention is due to use of methylprednisolone during the incubationstage of the cell preparation's production process. According to someembodiments, methylprednisolone is used in the present invention duringproduction of the cell preparation of the invention, as part of theprocedure in which the cells are induced to enter an early-apoptoticstate.

According to some embodiments, the composition of the invention furthercomprises methylprednisolone at a concentration between 0.5-30 μg/ml,possibly 1-25 μg/ml, typically between 3-22 μg/ml. Each possibilityrepresents a separate embodiment of the present invention. According tosome embodiments, the composition of the invention comprisesmethylprednisolone at a concentration between 3.7-21.9 μg/ml.

According to some embodiments, the composition of the invention furthercomprises methylprednisolone at a concentration that does not exceed 30μg/ml. According to some embodiments, the composition of the inventionfurther comprises methylprednisolone at a concentration that does notexceed 30 μg/ml, possibly does not exceed 25 μg/ml, typically does notexceed 21.9 μg/ml. Each possibility represents a separate embodiment ofthe present invention.

According to some embodiments, the composition of the invention furthercomprises methylprednisolone at a concentration between 0.5-60 μg/ml,possibly 1.12-60 μg/ml. Each possibility represents a separateembodiment of the present invention. According to some embodiments, thecomposition of the invention further comprises methylprednisolone at aconcentration that does not exceed 60 μg/ml.

According to some embodiments, the composition of the inventioncomprises at least 0.5 μg/ml, possibly at least 1 μg/ml, alternativelyat least 3 μg/ml methylprednisolone. Each possibility represents aseparate embodiment of the present invention. According to someembodiments, the composition of the invention comprises at least 3.5μg/ml methylprednisolone. According to some embodiments, the compositionof the invention comprises at least 3.7 μm/ml methylprednisolone.

According to some embodiments, the composition of the invention furthercomprises between 0.1-25 mg methylprednisolone, possibly between 0.4-20mg methylprednisolone, alternatively between 0.67-18 mgmethylprednisolone. Each possibility represents a separate embodiment ofthe present invention. According to some embodiments, the composition ofthe invention further comprises methylprednisolone in an amount thatdoes not exceed 25 mg, typically 20 mg, alternatively 18 mg. Eachpossibility represents a separate embodiment of the present invention.According to certain embodiments, the composition of the inventionfurther comprises methylprednisolone in an amount that does not exceed15 mg.

According to some embodiments, the pharmaceutical composition of theinvention comprises a cell preparation comprising mononuclear enrichedcells, wherein the preparation comprises at least 85% mononuclear cells,wherein at least 40% of the cells in the preparation are in an earlyapoptotic state, wherein at least 85% of the cells in the preparationare viable cells and wherein the preparation comprises no more than 15%polymorphonuclear leukocytes. According to certain embodiments, thepharmaceutical composition of the invention comprises a cell preparationcomprising mononuclear enriched cells, wherein the preparation comprisesat least 85% mononuclear cells, wherein at least 40% of the cells in thepreparation are in an early apoptotic state and wherein at least 85% ofthe cells in the preparation are viable cells.

According to some embodiments, the pharmaceutical composition of theinvention comprises a cell preparation comprising mononuclear enrichedcells, wherein the preparation comprises at least 85% mononuclear cells,wherein at least 40% of the cells in the preparation are in an earlyapoptotic state, wherein at least 85% of the cells in the preparationare viable cells and wherein the pharmaceutical composition comprises ananti-coagulant. According to some embodiments, the anti-coagulant isselected from the group consisting of: heparin, ACD Formula A and acombination thereof. Each possibility represents a separate embodimentof the present invention.

According to some embodiments, the pharmaceutical composition of theinvention comprises a cell preparation comprising mononuclear enrichedcells, wherein the preparation comprises at least 85% mononuclear cells,wherein at least 40% of the cells in the preparation are in an earlyapoptotic state, wherein at least 85% of the cells in the preparationare viable cells, wherein the preparation comprises no more than 15%polymorphonuclear leukocytes and wherein the pharmaceutical compositioncomprises an anti-coagulant. According to some embodiments, theanti-coagulant is selected from the group consisting of: heparin, ACDFormula A and a combination thereof. Each possibility represents aseparate embodiment of the present invention.

According to some embodiments, the heparin in the pharmaceuticalcomposition is present at a concentration between 0.005 U/ml and 2.5U/ml. According to other embodiments, the ACD Formula A in thepharmaceutical composition is present at a concentration of 0.01%-10%v/v, alternatively 0.05%-5% v/v. Each possibility represents a separateembodiment of the present invention.

According to some embodiments, the pharmaceutical composition of theinvention comprises a cell preparation comprising mononuclear enrichedcells, wherein the preparation comprises at least 85% mononuclear cells,wherein at least 40% of the cells in the preparation are in an earlyapoptotic state, wherein at least 85% of the cells in the preparationare viable cells, wherein the preparation comprises no more than 15%polymorphonuclear leukocytes and wherein the preparation comprisesmethylprednisolone at a concentration which does not exceed 30 μg/ml.According to certain embodiments, the pharmaceutical composition of theinvention comprises a cell preparation comprising mononuclear enrichedcells, wherein the preparation comprises at least 85% mononuclear cells,wherein at least 40% of the cells in the preparation are in an earlyapoptotic state, wherein at least 85% of the cells in the preparationare viable cells and wherein the preparation comprisesmethylprednisolone at a concentration which does not exceed 30 μg/ml.

According to some embodiments, the pharmaceutical composition of theinvention comprises a cell preparation comprising mononuclear enrichedcells, wherein the preparation comprises at least 85% mononuclear cells,wherein at least 40% of the cells in the preparation are in an earlyapoptotic state, wherein at least 85% of the cells in the preparationare viable cells, wherein the preparation comprises no more than 15%polymorphonuclear leukocytes and wherein the preparation comprises ananti-coagulant and methylprednisolone. According to some embodiment, theconcentration of methylprednisolone in the pharmaceutical composition ofthe invention does not exceed 30 μg/ml. According to certainembodiments, the pharmaceutical composition of the invention comprises acell preparation comprising mononuclear enriched cells, wherein thepreparation comprises at least 85% mononuclear cells, wherein at least40% of the cells in the preparation are in an early apoptotic state,wherein at least 85% of the cells in the preparation are viable cellsand wherein the preparation comprises an anti-coagulant andmethylprednisolone. According to some embodiment, the concentration ofmethylprednisolone in the pharmaceutical composition of the inventiondoes not exceed 30 μg/ml.

In particular embodiments, the pharmaceutical composition of theinvention is administered at a dosage of about 30×10⁶-300×10⁶ cells perkg body weight, 100×10⁶-300×10⁶ cells per kg body weight, alternativelyabout 120×10⁶-250×10⁶ cells per kg body weight. Each possibilityrepresents a separate embodiment of the present invention. In particularembodiments, the pharmaceutical composition of the invention isadministered at a dosage of about 35×10⁶ cells per kg body weight.According to some embodiments, the pharmaceutical composition of theinvention is administered at a dosage of about 140×10⁶-210×10⁶ cells perkg body weight. According to a particular embodiment, the pharmaceuticalcomposition of the invention is administered at a dosage of about140×10⁶ cells per kg body weight. According to another particularembodiment, the pharmaceutical composition of the invention isadministered at a dosage of about 210×10⁶ cells per kg body weight.According to another particular embodiment, the pharmaceuticalcomposition of the invention is administered at a dosage of about35×10⁶-210×10⁶ cells per kg body weight. According to another particularembodiment, the pharmaceutical composition of the invention isadministered at a dosage of about 250×10⁶ cells per kg body weight. Inother embodiments, the pharmaceutical composition of the invention isadministered at a dosage of about 5×10⁶ cells per kg body weight. Itshould be appreciated that said low dosage is suitable for localinjection of the compositions disclosed herein, such as local injectionto a joint for treating arthritis.

According to some embodiments, the therapeutic mononuclear-enriched cellpreparation of the invention is administered to the subjectsystemically, preferably via the intravenous route. Each possibilityrepresents a separate embodiment of the present invention. Alternately,the therapeutic mononuclear enriched cell may be administered to thesubject according to various other routes, including, but not limitedto, the parenteral, intraperitoneal, intra-articular, intramuscular andsubcutaneous routes. Each possibility represents a separate embodimentof the present invention. Preferably, the therapeutic mononuclearenriched cells are administered to the subject suspended in a suitablephysiological buffer, such as, but not limited to, saline solution, PBS,HBSS, and the like. In addition the suspension medium may furthercomprise supplements conducive to maintaining the viability of thecells.

Methods of Producing Apoptotic Cell Preparations

According to another aspect, the present invention provides a method forproducing the pharmaceutical composition of the invention (referred toherein as “the production method of the invention”), wherein the methodcomprises:

obtaining a mononuclear-enriched cell composition from the peripheralblood of a donor, the mononuclear-enriched cell composition comprisingat least 65% mononuclear cells;

freezing the mononuclear-enriched cell composition in a freezing medium;

thawing the mononuclear-enriched cell composition;

incubating the mononuclear-enriched cell composition in an incubationmedium comprising methylprednisolone at a final concentration of about10-100 μg/mL;

wherein at least one of the freezing medium and the incubation mediumcomprise an anti-coagulant; and

suspending said cell composition in an administration medium, therebyproviding the pharmaceutical composition of the invention. Eachpossibility represents a separate embodiment of the present invention.

According to some embodiments, the pharmaceutical composition obtainedaccording to the production method of the invention comprises at least85% mononuclear cells. In further embodiments, the pharmaceuticalcomposition contains at least 85% mononuclear cells, 90% mononuclearcells or alternatively over 90% mononuclear cells. Each possibility is aseparate embodiment of the invention. According to some embodiments, thepharmaceutical composition comprises at least 90% mononuclear cells.According to some embodiments, the pharmaceutical composition comprisesat least 95% mononuclear cells.

According to some embodiments, obtaining a mononuclear-enriched cellcomposition according to the production method of the invention iseffected by leukapheresis. As used herein, the term “leukapheresis”refers to an apheresis procedure in which leukocytes are separated fromthe blood of a donor. According to some embodiments, the blood of adonor undergoes leukapheresis and thus a mononuclear-enriched cellcomposition is obtained according to the production method of theinvention. It is to be noted, that the use of at least one anticoagulantduring leukapheresis is required, as is known in the art, in order toprevent clotting of the collected cells.

According to some embodiments, the leukapheresis procedure is configuredto allow collection of mononuclear-enriched cell composition accordingto the production method of the invention. According to someembodiments, cell collections obtained by leukapheresis comprise atleast 65%, preferably at least 70%, most preferably at least 80%mononuclear cells. Each possibility represents a separate embodiment ofthe present invention. According to some embodiments, blood plasma fromthe cell-donor is collected in parallel to obtaining of themononuclear-enriched cell composition according to the production methodof the invention. According to some embodiments, about 300-600 ml ofblood plasma from the cell-donor are collected in parallel to obtainingthe mononuclear-enriched cell composition according to the productionmethod of the invention. According to some embodiments, blood plasmacollected in parallel to obtaining the mononuclear-enriched cellcomposition according to the production method of the invention is usedas part of the freezing and/or incubation medium. Each possibilityrepresents a separate embodiment of the present invention.

It is to be noted that, according to some embodiments, while themononuclear-enriched cell preparation at cell collection comprises atleast 65%, preferably at least 70%, most preferably at least 80%mononuclear cells, the final pharmaceutical composition of theinvention, following the production method of the invention, comprisesat least 85%, preferably at least 90%, most preferably at least 95%mononuclear cells. Each possibility represents a separate embodiment ofthe present invention.

According to certain embodiments, the mononuclear-enriched cellpreparation used for production of the composition of the inventioncomprises at least 50% mononuclear cells at cell collection. Accordingto certain embodiments, the present invention provides a method forproducing the pharmaceutical composition of the invention wherein themethod comprises obtaining a mononuclear-enriched cell preparation fromthe peripheral blood of a donor, the mononuclear-enriched cellpreparation comprising at least 50% mononuclear cells. According tocertain embodiments, the present invention provides a method forproducing the pharmaceutical composition of the invention wherein themethod comprises freezing a mononuclear-enriched cell preparationcomprising at least 50% mononuclear cells.

According to some embodiments, the mononuclear-enriched cell compositionobtained according to the production method of the invention undergoesfreezing in a freezing medium. According to some embodiments, thefreezing is gradual. According to some embodiments, following collectionthe cells are maintained at room temperature until frozen. According tosome embodiments, the cell-preparation undergoes at least one washingstep in washing medium following cell-collection and prior to freezing.As used herein, the terms “obtaining cells” and “cell collection” areused interchangeably. According to some embodiments, the cells of thecell preparation of the invention are frozen within 3-6 hours ofcollection. According to some embodiments, the cell preparation of theinvention is frozen within up to 6 hours of cell collection. Accordingto some embodiments, the cells of the cell preparation of the inventionare frozen within 1, 2, 3, 4, 5, 6, 7, 8 hours of collection. Eachpossibility represents a separate embodiment of the present invention.According to other embodiments, the cells of the cell preparation of theinvention are frozen up to 8, 12, 24, 48, 72 hours of collection. Eachpossibility represents a separate embodiment of the present invention.According to other embodiments, following collection the cells aremaintained at 2-8° C. until frozen.

According to some embodiments, freezing according to the productionmethod of the invention comprises: freezing the cell preparation atabout −18° C. to −25° C. followed by freezing the cell preparation atabout −80° C. and finally freezing the cell preparation in liquidnitrogen until thawing. According to some embodiments, the freezingaccording to the production method of the invention comprises: freezingthe cell preparation at about −18° C. to −25° C. for at least 2 hours,freezing the cell preparation at about −80° C. for at least 2 hours andfinally freezing the cell preparation in liquid nitrogen until thawing.According to some embodiments, the cells are kept in liquid nitrogen forat least 8, 10 or 12 hours prior to thawing. Each possibility representsa separate embodiment of the present invention. According to someembodiments, the cells of the cell preparation are kept in liquidnitrogen until thawing and incubation with apoptosis-inducing incubationmedium. According to some embodiments, the cells of the cell preparationare kept in liquid nitrogen until the day of hematopoietic stem celltransplantation. According to non-limiting examples, the time from cellcollection and freezing to preparation of the final composition of theinvention may be between 1-50 days, alternatively between 6-30 days.Each possibility represents a separate embodiment of the presentinvention. According to alternative embodiments, the cell preparationmay be kept in liquid nitrogen for longer time periods, such as at leastseveral months.

According to some embodiments, the freezing according to the productionmethod of the invention comprises freezing the cell preparation at about−18° C. to −25° C. for at least 0.5, 1, 2, 4 hours. Each possibilityrepresents a separate embodiment of the present invention. According tosome embodiments, the freezing according to the production method of theinvention comprises freezing the cell preparation at about −18° C. to−25° C. for about 2 hours. According to some embodiments, the freezingaccording to the production method of the invention comprises freezingthe cell preparation at about −80° C. for at least 0.5, 1, 2, 4, 12hours. Each possibility represents a separate embodiment of the presentinvention.

According to some embodiments, the mononuclear-enriched cell compositionmay remain frozen at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 20months. Each possibility represents a separate embodiment of the presentinvention. According to some embodiments, the mononuclear-enriched cellcomposition may remain frozen at least 0.5, 1, 2, 3, 4, 5 years. Eachpossibility represents a separate embodiment of the present invention.According to certain embodiments, the mononuclear-enriched cellcomposition may remain frozen for at least 20 months.

According to some embodiments, the mononuclear-enriched cell compositionis frozen for at least 8, 10, 12, 18, 24 hours. Each possibilityrepresents a separate embodiment of the present invention. According tocertain embodiments, freezing the mononuclear-enriched cell compositionis for a period of at least 8 hours. According to some embodiments, themononuclear-enriched cell composition is frozen for at least about 10hours. According to some embodiments, the mononuclear-enriched cellcomposition is frozen for at least about 12 hours. According to someembodiments, the mononuclear-enriched cell composition is frozen forabout 12 hours. According to some embodiments, the total freezing timeof the mononuclear-enriched cell composition (at about −18° C. to −25°C., at about −80° C. and in liquid nitrogen) is at least 8, 10, 12, 18,24 hours. Each possibility represents a separate embodiment of thepresent invention.

According to some embodiments, the freezing at least partly induces theearly-apoptotic state in the cells of the mononuclear-enriched cellcomposition. According to some embodiments, the freezing mediumcomprises RPMI 1640 medium comprising L-glutamine, Hepes, Hes, dimethylsulfoxide (DMSO) and plasma. According to some embodiments, the plasmain the freezing medium is an autologous plasma of the donor whichdonated the mononuclear-enriched cells of the composition of theinvention. According to some embodiments, the freezing medium comprisesRPMI 1640 medium comprising 2 mM L-glutamine, 10 mM Hepes, 5% Hes, 10%dimethyl sulfoxide and 20% v/v plasma.

According to some embodiments, the freezing medium comprises ananti-coagulant. According to certain embodiments, at least some of themedia used during the production method of the invention, including thefreezing medium, the incubation medium and the washing media comprise ananti-coagulant. According to certain embodiments, all media used duringthe production method of the invention which comprise an anti-coagulantcomprise the same concentration of anti-coagulant. According to someembodiments, anti-coagulant is not added to the final suspension mediumof the cell composition of the invention.

According to some embodiments, addition of an anti-coagulant at least tothe freezing medium improves the yield of the cell-preparation of theinvention. According to other embodiments, addition of an anti-coagulantto the freezing medium improves the yield of the cell-preparation in thepresence of a high triglyceride level. As used herein, improvement inthe yield of the cell-preparation of the invention relates toimprovement in at least one of: the percentage of viable cells out ofcells frozen, the percentage of early-state apoptotic cells out ofviable cells and a combination thereof. Each possibility represents aseparate embodiment of the present invention.

According to some embodiments, cell yield in the composition of theinvention relates to cell number in the composition out of the initialnumber of cells subjected to apoptosis induction according to thepresent invention. As used herein, the terms “induction of earlyapoptotic state” and “induction of apoptosis” are used interchangeably.

According to some embodiments, improvement in the yield of thecell-preparation of the invention relates to improvement in the numberof the early-apoptotic viable cells of the preparation out of the numberof frozen cells from which the preparation was produced.

According to some embodiments, addition of an anti-coagulant to thefreezing medium contributes to a high and stable yield between differentpreparations of the pharmaceutical composition of the invention.According to preferable embodiments, addition of an anti-coagulant atleast to the freezing medium and incubation medium results in a high andstable yield between different preparations of the pharmaceuticalcomposition, regardless to the cell collection protocol used.

According to some embodiments, the freezing medium comprises ananti-coagulant selected from the group consisting of: heparin, ACDFormula A and a combination thereof. Each possibility represents aseparate embodiment of the present invention. According to someembodiments, the anti-coagulant used in the freezing medium is ACDFormula A containing heparin at a concentration of 10 U/ml. According tosome embodiments, the freezing medium comprises 5% v/v of ACD Formula Asolution comprising heparin at a concentration of 10 U/ml.

According to some embodiments, the freezing medium comprises heparin.According to some embodiments, the heparin in the freezing medium is ata concentration of between 0.1-2.5 U/ml. According to some embodiments,the heparin in the freezing medium is at a concentration of between0.1-2.5 U/ml, possibly between 0.3-0.7 U/ml, typically about 0.5 U/ml.Each possibility represents a separate embodiment of the presentinvention. According to certain embodiments, the heparin in the freezingmedium is at a concentration of about 0.5 U/ml.

According to some embodiments, the freezing medium comprises ACD FormulaA. According to some embodiments, the ACD Formula A in the freezingmedium is at a concentration of between 1%-15% v/v. According to someembodiments, the ACD Formula A in the freezing medium is at aconcentration of between 1%-15% v/v, possibly between 4%-7% v/v,typically about 5% v/v. Each possibility represents a separateembodiment of the present invention. According to some embodiments, theACD Formula A in the freezing medium is at a concentration of about 5%v/v.

According to some embodiments, the mononuclear-enriched cell compositionundergoes at least one washing step following cell collection and priorto being re-suspended in the freezing medium and frozen. According tosome embodiments, the mononuclear-enriched cell composition undergoes atleast one washing step following freezing and thawing. According to someembodiments, washing steps comprise centrifugation of themononuclear-enriched cell composition followed by supernatant extractionand re-suspension in washing medium.

According to some embodiments, cell collection refers to obtaining amononuclear-enriched cell composition. According to some embodiments,washing steps performed during the production method of the inventionare performed in a washing medium. According to certain embodiments,washing steps performed up until the incubation step of the productionmethod of the invention are performed in a washing medium. According tosome embodiments, the washing medium comprises RPMI 1640 mediumsupplemented with L-glutamine and Hepes. According to some embodiments,the washing medium comprises RPMI 1640 medium supplemented with 2 mML-glutamine and 10 mM Hepes.

According to some embodiments, the washing medium comprises ananti-coagulant. According to some embodiments, the washing mediumcomprises an anti-coagulant selected from the group consisting of:heparin, ACD Formula A and a combination thereof. Each possibilityrepresents a separate embodiment of the present invention. According tosome embodiments, the concentration of the anti-coagulant in the washingmedium is the same concentration as in the freezing medium. According tosome embodiments, the concentration of the anti-coagulant in the washingmedium is the same concentration as in the incubation medium. Accordingto some embodiments, the anti-coagulant used in the washing medium isACD Formula A containing heparin at a concentration of 10 U/ml.

According to some embodiments, the washing medium comprises heparin.According to some embodiments, the heparin in the washing medium is at aconcentration of between 0.1-2.5 U/ml. According to some embodiments,the heparin in the washing medium is at a concentration of between0.1-2.5 U/ml, possibly between 0.3-0.7 U/ml, typically about 0.5 U/ml.Each possibility represents a separate embodiment of the presentinvention. According to certain embodiments, the heparin in the washingmedium is at a concentration of about 0.5 U/ml.

According to some embodiments, the washing medium comprises ACD FormulaA. According to some embodiments, the ACD Formula A in the washingmedium is at a concentration of between 1%-15% v/v. According to someembodiments, the ACD Formula A in the washing medium is at aconcentration of between 1%-15% v/v, possibly between 4%-7% v/v,typically about 5% v/v. Each possibility represents a separateembodiment of the present invention. According to some embodiments, theACD Formula A in the washing medium is at a concentration of about 5%v/v.

According to some embodiments, the mononuclear-enriched cell compositionis thawed several hours prior to the intended administration of thecomposition of the invention to a subject. According to someembodiments, the mononuclear-enriched cell composition is thawed atabout 33° C.-39° C. According to some embodiments, themononuclear-enriched cell composition is thawed for about 30-240seconds, preferably 40-180 seconds, most preferably 50-120 seconds. Eachpossibility represents a separate embodiment of the present invention.

According to some embodiments, the mononuclear-enriched cell compositionis thawed at least 10 hours prior to the intended administration of thecomposition of the invention, alternatively at least 20, 30, 40 or 50hours prior to the intended administration of the composition of theinvention. Each possibility represents a separate embodiment of thepresent invention. According to some embodiments, themononuclear-enriched cell composition is thawed at least 15-24 hoursprior to the intended administration of the composition of theinvention. According to some embodiments, the mononuclear-enriched cellcomposition is thawed at least about 24 hours prior to the intendedadministration of the composition of the invention. According to someembodiments, the mononuclear-enriched cell composition is thawed atleast 20 hours prior to the intended administration of the compositionof the invention. According to some embodiments, themononuclear-enriched cell composition is thawed 30 hours prior to theintended administration of the composition of the invention. Accordingto some embodiments, the mononuclear-enriched cell composition is thawedat least 24 hours prior to the intended administration of thecomposition of the invention. According to some embodiments, themononuclear-enriched cell composition undergoes at least one step ofwashing in the washing medium before and/or after thawing. Eachpossibility represents a separate embodiment of the present invention.

According to some embodiments, the mononuclear-enriched cell compositionis incubated in incubation medium following freezing and thawing.According to some embodiments, there is at least one washing stepbetween thawing and incubation. As used herein, the terms “incubationmedium” and “apoptosis inducing incubation medium” are usedinterchangeably. According to some embodiments, the incubation mediumcomprises RPMI 1640 medium supplemented with L-glutamine, Hepesmethylprednisolone and plasma. According to some embodiments, thewashing medium comprises 2 mM L-glutamine, 10 mM Hepes and 10% v/v bloodplasma. According to some embodiments, the blood plasma in theincubation medium is derived from the same donor from whom the cells ofthe cell preparation of the invention are derived. According to someembodiments, the blood plasma is added to the incubation medium on theday of incubation. According to some embodiments, incubation isperformed at 37° C. and 5% CO₂.

According to some embodiments, the incubation medium comprisesmethylprednisolone. According to some embodiments, themethylprednisolone within the incubation medium further induces thecells in the mononuclear-enriched cell composition to enter anearly-apoptotic state. According to some embodiments, the cells in themononuclear-enriched cell composition are induced to enter anearly-apoptotic state both by freezing and incubating in the presence ofmethylprednisolone. According to some embodiments, the production methodof the invention advantageously allows induction of an early-apoptosisstate substantially without induction of necrosis, wherein the cellsremain stable at said early-apoptotic state for about 24 hours followingpreparation.

According to some embodiments, the incubation medium comprisesmethylprednisolone at a concentration of about 10-100 μg/ml. Accordingto some embodiments, the incubation medium comprises methylprednisoloneat a concentration of about 40-60 μg/ml, alternatively about 45-55μg/ml. Each possibility represents a separate embodiment of the presentinvention. According to some embodiments, the incubation mediumcomprises methylprednisolone at a concentration of 50 μg/ml.

According to some embodiments, the incubation is for about 2-12 hours,possibly 4-8 hours, typically for about 5-7 hours. Each possibilityrepresents a separate embodiment of the present invention. According tosome embodiments, the incubation is for about 6 hours. According to someembodiments, the incubation is for at least 6 hours. According to apreferred embodiment, the incubation is for 6 hours.

According to some embodiments, the incubation medium comprises ananti-coagulant. According to some embodiments, addition of ananti-coagulant to the incubation medium improves the yield of thecell-preparation of the invention. According to some embodiments, theanti-coagulant in the incubation medium is of the same concentration aswithin the freezing medium. According to some embodiments, theincubation medium comprises an anti-coagulant selected from the groupconsisting of: heparin, ACD Formula A and a combination thereof. Eachpossibility represents a separate embodiment of the present invention.According to some embodiments, the anti-coagulant used in the incubationmedium is ACD Formula A containing heparin at a concentration of 10U/ml.

According to some embodiments, the incubation medium comprises heparin.According to some embodiments, the heparin in the incubation medium isat a concentration of between 0.1-2.5 U/ml. According to someembodiments, the heparin in the incubation medium is at a concentrationof between 0.1-2.5 U/ml, possibly between 0.3-0.7 U/ml, typically about0.5 U/ml. Each possibility represents a separate embodiment of thepresent invention. According to certain embodiments, the heparin in theincubation medium is at a concentration of about 0.5 U/ml.

According to some embodiments, the incubation medium comprises ACDFormula A. According to some embodiments, the ACD Formula A in theincubation medium is at a concentration of between 1%-15% v/v. Accordingto some embodiments, the ACD Formula A in the incubation medium is at aconcentration of between 1%-15% v/v, possibly between 4%-7% v/v,typically about 5% v/v. Each possibility represents a separateembodiment of the present invention. According to some embodiments, theACD Formula A in the incubation medium is at a concentration of about 5%v/v.

According to some embodiments, both the freezing medium and theincubation medium comprise an anti-coagulant. According to someembodiments, addition of an anti-coagulant both to the incubation mediumand freezing medium results in a high and stable cell-yield betweendifferent preparations of the composition of the invention regardless ofcell-collection conditions, such as, but not limited to, the timingand/or type of anti-coagulant added during cell collection. According tosome embodiments, addition of an anti-coagulant both to the incubationmedium and freezing medium results in a high and stable yield of thecell-preparation of the invention regardless of the timing and/or typeof anti-coagulant added during leukapheresis. According to someembodiments, production of the cell-preparation of the invention in thepresence of a high triglyceride level results in a low and/or unstablecell-yield between different preparations. Each possibility represents aseparate embodiment of the present invention. According to someembodiments, producing the cell-preparation from the blood of a donorhaving a high triglyceride level results in a low and/or unstablecell-yield of the cell preparation. Each possibility represents aseparate embodiment of the present invention. According to someembodiments, the term “high triglyceride level” refers to a triglyceridelevel which is above the normal level of a healthy subject of the samesex and age. According to some embodiments, the term “high triglyceridelevel” refers to a triglyceride level above about 1.7 milimole/liter. Asused herein, a high and stable yield refers to a cell yield in thecomposition of the invention which is high enough to enable preparationof a dose which will demonstrate therapeutic efficiency whenadministered to a subject. According to some embodiments, therapeuticefficiency refers to the ability to treat, prevent or ameliorate animmune disease, an autoimmune disease or an inflammatory disease in asubject. Each possibility represents a separate embodiment of thepresent invention. According to some embodiments, a high and stable cellyield is a cell yield of at least 30%, possibly at least 40%, typicallyat least 50% of cells in the composition of the invention out of cellsinitially frozen. Each possibility represents a separate embodiment ofthe present invention.

According to some embodiments, in case the cell-preparation of theinvention is obtained from a donor having a high triglyceride level, thedonor will take at least one measure selected from the group consistingof: taking triglyceride-lowering medication prior to donation, such as,but not limited to: statins and/or bezafibrate, fasting for a period ofat least 8, 10, 12 hours prior to donation, eating an appropriate dietto reduce blood triglyceride level at least 24, 48, 72 hours prior todonating and any combination thereof. Each possibility represents aseparate embodiment of the present invention.

According to some embodiments, addition of an anti-coagulant to theincubation medium and/or freezing medium results in a high and stablecell yield within the composition of the invention regardless of thetriglyceride level in the blood of the donor. According to someembodiments, addition of an anti-coagulant to the incubation mediumand/or freezing medium results in a high and stable cell yield withinthe composition the invention when obtained from the blood of a donorhaving normal or high triglyceride level. According to some embodiments,addition of an anti-coagulant at least to the incubation medium, resultsin a high and stable cell yield within the composition of the inventionregardless of the triglyceride level in the blood of the donor.According to some embodiments, addition of an anti-coagulant to thefreezing medium and incubation medium results in a high and stable cellyield within the composition of the invention regardless of thetriglyceride level in the blood of the donor.

According to some embodiments, the freezing medium and/or incubationmedium and/or washing medium comprise heparin at a concentration of atleast 0.1 U/ml, possibly at least 0.3 U/ml, typically at least 0.5 U/ml.Each possibility represents a separate embodiment of the presentinvention. According to some embodiments, the freezing medium and/orincubation medium and/or washing medium comprise ACD Formula A at aconcentration of at least 1% v/v, possibly at least 3% v/v, typically atleast 5% v/v. Each possibility represents a separate embodiment of thepresent invention.

According to some embodiments, the mononuclear-enriched cell compositionundergoes at least one washing step between each stage of the productionmethod of the invention. According to some embodiments, anti-coagulantis added to washing media during washing steps throughout the productionmethod of the invention. According to some embodiments, themononuclear-enriched cell composition undergoes at least one washingstep following incubation. According to some embodiments, themononuclear-enriched cell composition undergoes at least one washingstep following incubation using PBS. According to some embodiments,anti-coagulant is not added to the final washing step prior tore-suspension of the cell-preparation in the administration medium.According to some embodiments, anti-coagulant is not added to the PBSused in the final washing step prior to re-suspension of thecell-preparation in the administration medium. According to certainembodiments, anti-coagulant is not added to the administration medium.

According to some embodiments, the cell concentration during incubatingis about 5×10⁶ cells/ml.

According to some embodiments, the mononuclear-enriched cell compositionis suspended in an administration medium following freezing, thawing andincubating, thereby resulting in the pharmaceutical composition of theinvention. According to some embodiments, the administration mediumcomprises a suitable physiological buffer. Non-limiting examples of asuitable physiological buffer are: saline solution, Phosphate BufferedSaline (PBS), Hank's Balanced Salt Solution (HBSS), and the like.According to some embodiments, the administration medium comprises PBS.According to some embodiments, the administration medium comprisessupplements conducive to maintaining the viability of the cells.According to some embodiments, the mononuclear-enriched cell compositionis filtered prior to administration. According to some embodiments, themononuclear-enriched cell composition is filtered prior toadministration using a filter of at least 200 μm.

According to some embodiments, the mononuclear-enriched cell compositionis re-suspended in an administration medium such that the final volumeof the resulting cell-preparation is between 100-1000 ml, possiblybetween 200-800 ml, typically between 300-600 ml. Each possibilityrepresents a separate embodiment of the present invention.

According to some embodiments, the present invention provides a methodfor producing the pharmaceutical composition of the invention, whereinthe method comprises:

freezing a mononuclear-enriched cell composition comprising at least 65%mononuclear cells in a freezing medium;

thawing the mononuclear-enriched cell composition;

incubating the mononuclear-enriched cell composition in an incubationmedium comprising methylprednisolone at a final concentration of about10-100 μg/mL;

wherein at least one of the freezing medium and the incubation mediumcomprise an anti-coagulant; and

reconstituting said cell composition in an administration medium,thereby providing the pharmaceutical composition of the invention. Eachpossibility represents a separate embodiment of the present invention.

According to some embodiments, the method for producing thepharmaceutical composition of the invention further comprises obtainingthe mononuclear-enriched cell composition. According to someembodiments, obtaining a mononuclear-enriched cell composition is fromthe peripheral blood of a donor. According to some embodiments,obtaining a mononuclear-enriched cell composition is from the peripheralblood of the same donor used for transplantation. According to someembodiments, obtaining a mononuclear-enriched cell composition is fromthe peripheral blood of the same donor donating cells for HSCT.According to some embodiments, obtaining a mononuclear-enriched cellcomposition for the production method of the invention is from a donorwhich is not undergoing hematopoietic stem-cells mobilization at thetime of obtaining According to some embodiments, obtaining amononuclear-enriched cell composition for the production method of theinvention is from a donor which is not undergoing treatment withGranulocyte Colony Stimulating Factor (G-CSF) at the time of obtaining.

According to some embodiments, the present invention provides a methodfor producing the pharmaceutical composition of the invention, whereinthe method comprises:

obtaining a mononuclear-enriched cell composition from the peripheralblood of a donor, said mononuclear-enriched cell composition comprisingat least 65% mononuclear cells;

freezing the mononuclear-enriched cell composition in a freezing medium,wherein said freezing medium comprises an anti-coagulant;

thawing the mononuclear-enriched cell composition;

incubating the mononuclear-enriched cell composition in an incubationmedium, wherein said incubation medium comprises an anti-coagulant andmethylprednisolone at a final concentration of about 50 μg/mL;

and

suspending said cell composition in an administration medium, therebyproviding the pharmaceutical composition of the invention.

According to some embodiments, the present invention provides thecell-preparation of the invention, wherein the cell-preparation isproduced by the production method of the invention.

According to some embodiments, the present invention provides apharmaceutical composition comprising a cell preparation comprisingmononuclear enriched cells, wherein the preparation comprises at least85% mononuclear cells, wherein at least 40% of the cells in thepreparation are in an early apoptotic state and wherein at least 85% ofthe cells in the preparation are viable cells; wherein saidpharmaceutical composition is produced by a method comprising:

obtaining a mononuclear-enriched cell composition from the peripheralblood of a donor, said mononuclear-enriched cell composition comprisingat least 65% mononuclear cells;

freezing the mononuclear-enriched cell composition in a freezing medium;

thawing the mononuclear-enriched cell composition;

incubating the mononuclear-enriched cell composition in an incubationmedium comprising methylprednisolone at a final concentration of about10-100 μg/mL;

wherein at least one of the freezing medium and the incubation mediumcomprise an anti-coagulant; and

suspending said cell composition in an administration medium, therebyproviding the composition of the invention. Each possibility representsa separate embodiment of the present invention.

According to some embodiments, the present invention provides apharmaceutical composition comprising a cell preparation comprisingmononuclear enriched cells, wherein the preparation comprises at least85% mononuclear cells, wherein at least 40% of the cells in thepreparation are in an early apoptotic state and wherein at least 85% ofthe cells in the preparation are viable cells; wherein saidpharmaceutical composition is produced by a method comprising:

obtaining a mononuclear-enriched cell composition from the peripheralblood of a donor, said mononuclear-enriched cell composition comprisingat least 65% mononuclear cells;

freezing the mononuclear-enriched cell composition in a freezing mediumcomprising an anti-coagulant;

thawing the mononuclear-enriched cell composition;

incubating the mononuclear-enriched cell composition in an incubationmedium comprising an anti-coagulant and methylprednisolone at a finalconcentration of about 50 μg/mL; and

suspending said cell composition in an administration medium, therebyproviding the composition of the invention. Each possibility representsa separate embodiment of the present invention.

According to some embodiments, the present invention provides apharmaceutical composition comprising a cell preparation comprisingmononuclear enriched cells, wherein the preparation comprises at least85% mononuclear cells, wherein at least 40% of the cells in thepreparation are in an early apoptotic state, wherein at least 85% of thecells in the preparation are viable cells and wherein the preparationcomprises no more than 15% polymorphonuclear leukocytes; wherein saidpharmaceutical composition is produced by a method comprising:

obtaining a mononuclear-enriched cell composition from the peripheralblood of a donor, said mononuclear-enriched cell composition comprisingat least 65% mononuclear cells;

freezing the mononuclear-enriched cell composition in a freezing medium;

thawing the mononuclear-enriched cell composition;

incubating the mononuclear-enriched cell composition in an incubationmedium comprising methylprednisolone at a final concentration of about10-100 μg/mL;

wherein at least one of the freezing medium and the incubation mediumcomprise an anti-coagulant; and

suspending said cell composition in an administration medium, therebyproviding the composition of the invention. Each possibility representsa separate embodiment of the present invention.

According to some embodiments, the present invention provides apharmaceutical composition comprising a cell preparation comprisingmononuclear enriched cells, wherein the preparation comprises at least85% mononuclear cells, wherein at least 40% of the cells in thepreparation are in an early apoptotic state, wherein at least 85% of thecells in the preparation are viable cells and wherein the preparationcomprises no more than 15% polymorphonuclear leukocytes; wherein saidpharmaceutical composition is produced by a method comprising:

obtaining a mononuclear-enriched cell composition from the peripheralblood of a donor, said mononuclear-enriched cell composition comprisingat least 65% mononuclear cells;

freezing the mononuclear-enriched cell composition in a freezing mediumcomprising an anti-coagulant;

thawing the mononuclear-enriched cell composition;

incubating the mononuclear-enriched cell composition in an incubationmedium comprising an anti-coagulant and methylprednisolone at a finalconcentration of about 50 μg/mL; and suspending said cell composition inan administration medium, thereby providing the composition of theinvention. Each possibility represents a separate embodiment of thepresent invention.

DEFINITIONS

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the terms “v/v” and “vol/vol” are used interchangeablyand refer to volume/volume concentration.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub ranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals there between.

As used herein the term “about” refers to plus/minus 10% of the valuestated.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

Preferably, the method of the present invention is used to treat thedisease in a mammalian subject, such as a human subject. It will bereadily appreciated that the method can be used to treat a human subjectin view of its successful clinical trial phase 1/2a as is describedherein.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub combination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

The following methods were employed in Examples 1-4 disclosed herein.

Analytical Methods and Specifications for ApoCell

ApoCell was tested during several stages of production. Quality controltests, test methods, testing facility, and specifications relating tothe collected mononuclear enriched cells prior to apoptosis inductionand the final ApoCell product preparation are described in Table 3below.

TABLE 3 Specifications for collected mononuclear-enriched cell fractionprior to freezing Specification Test Method Test At least 10⁹ totalcells SYSMEX Hematology Cell Count Analyzer At least 85% PI negativeFlow cytometric analysis of Cell viability cells propidium iodidestained cells At least 30% mononuclear SYSMEX Hematology Identity/puritycells Analyzer Negative growth at 5 and Direct sterility test Sterility14 days or equivalent Hy Laboratories Less than 1 EU/mL End safeEndotoxinApoCell was further tested prior to release for clinical administration.Quality control tests, test methods, testing facility, andspecifications for product release are presented in Table 4 below.Sterility and potency were performed after product release.

TABLE 4 Quality Control Test for Release of Product Specification TestMethod Test Per protocol Micros 60 Analyzer Cell number At least 85%viable Flow cytometric analysis Cell viability of propidium iodidestained cells At least 40% apoptotic Flow cytometric analysis Identity:cells of annexin V-propidium Apoptosis iodide Negative Hy LaboratoriesGram Stain Less than 1 EU/mL Endosafe Endotoxin CD15^(high) < 10% Flowcytometric analysis Identity/Purity of CD15 Negative growth at 14 daysDirect sterility test Sterility Hy Laboratories or equivalent SOP #10-004- 006 Inhibition of LPS Dendritic cell assay or Potencyupregulation of CD86 equivalent BR- or MHC class II PT01&PT02Cell Count

Cell count number is derived from the white blood cell (WBC) count.

Cell Viability Cell viability is determined by propidium iodide stainingby flow cytometry.

Identity/Purity

Identity and purity of the collected mononuclear enriched cells wasdetermined from the differential count performed on the SYSMEXhematology analyzer. The identity/purity was calculated by the sum oflymphocyte and monocyte percentage of WBC count. If mononuclearpercentage is less that 30% by hematology analyzer, an additionalevaluation method can be used—Flow Cytometry with specific identifyingantibodies: Double staining for CD15 and CD14 will be performed and thepercentage of granulocytes will be determined as a percentage ofCD15highCD14low-neg cells. If CD15highCD14low-neg cells portion will beless than 70% the collection will be allowed for further processing. Ifit is higher than 70%, the specific collection will be discarded.Alternatively equivalent test for granulocyte percentage determinationcan be used.

Identity/Purity prior to release of the ApoCell product was determinedby flow cytometric analysis to evaluate proportion of granulocytes.Double staining for CD15 and CD14 were performed and the percentage ofgranulocytes was determined as a percentage of CD15^(high)CD14^(low-neg)cells.

Identity: Apoptotic Phenotype

Apoptosis is determined by two-color flow cytometric assay to evaluatethe annexin and propidium iodide staining (An+PI−).

Sterility

Each lot of donor cells is tested for sterility using “direct sterilitytest method” with compliance to FDA regulation 21 CFR 610.12. Samplesare monitored daily, and readout results are documented at 14 postinoculation.

Endotoxin (LAL)

The Endosafe-PTS is an FDA-licensed endotoxin detection system thatutilizes an LAL test cartridge along with a handheld spectrophotometerto provide point-of-use results. The PTS provides quantitative LAL testresults in about 15 min.

Potency

Immature DC (iDCs) were prepared 6 days prior ApoCell preparation asdescribed in Verbovetski et al. (JEM 2002). Briefly, immaturemonocyte-derived dendritic cells, derived from a subject other than thedonor or recipient of the cell-preparation, were generated from theCD14+ selected fraction of blood buffy coats. PBMCs were isolated usingFicoll (GE Healthcare Life Sciences, Piscataway, N.J., US) and anti-CD14magnetic beads were used in order to isolate monocytes from PBMCsaccording to the manufacturer's instructions (BD Biosciences, San Jose,Calif., US). Monocytes were placed in wells at a concentration of1.25×10⁶/1.5 ml culture media in the presence of 1% autologous plasma,GMCSF (0.1 μg/ml), and IL-4 (0.1 μg/ml) (PeproTech, Rocky Hill, N.J.,US). Every 2 days 0.15 ml were removed and 0.25 ml media containingplasma, IL-4 (0.05 μg/ml), and GMCSF (0.1 μg/ml), were added. By day6, >90% of the cells were CD14−, with low expression of DR, CD-83, andCD-86. ApoCell was introduced to iDC at 1:2, 1:4, and 1:8 DCs:ApoCellratios overnight (16-24 hours). In some treatments, in order to evaluatethe anti-inflammatory effect, 10 ng/ml LPS were added 2 h followinginteraction. Following the interaction the cells were harvested andstained with both DCsign (for verifying the identity of iDC) and HLA-DRor CD86 (for evaluating the pro-inflammatory immune response). Isotypecontrols were used as controls. Expression of HLA-DR and CD86 ondendritic cells (DCsign positive cells) was evaluated using flowcytometry (FACSCalibur, Becton Dickenson, San Jose, Calif., USA).Analysis was performed on DCsing positive cells (10000 events) usingFCSexpress software.

Significant (Kolmogorov-Smirnov analysis) downregulation in at least oneof the markers in at least one ratio are used as a marker for tolerizingphenotype of DC following interaction with ApoCell.

Study Design and Patients

A multicenter phase 1/2a clinical trial of ApoCell (registered withclinicaltrials.gov; NO. NCT00524784) was performed in subjectsundergoing allogeneic sibling HLA-matched bone marrow transplantation.The study was performed to assess the safety tolerability andpreliminary efficacy of ApoCell administration.

The primary endpoint of the study was to determine the safety profileand tolerability (dose limiting toxicity (DLT)) of ascending doses ofApoCell in subjects undergoing allogeneic sibling HLA-matched bonemarrow transplantation within 180 days post-transplantation.

The secondary end points were to determine the success rate forallogeneic BMT (alloBMT) engraftment and time to successful engraftment,describe the rates and grade of acute GVHD following ApoCell infusionand determine the immunological function of the recipient followingApoCell infusion and alloBMT. In order to evaluate the outcomes thefollowing parameters were used: Time to neutrophil and plateletrecovery, time to full donor chimerism in neutrophils, proportions ofsubjects with graft failure, relapse or malignancy, incidence ofinfections (bacterial, viral and fungal), proportions of subjects withoverall survival at Day 45 (46 days after ApoCell infusion), at Day 100and at day 180, proportions of subjects with acute GvHD-free survival atDay 180, rates and grade of acute GvHD following ApoCell infusion,proportions of subjects who developed acute GvHD grade time to onset ofacute GvHD. Additional secondary end points included time to engraftmentand time to discharge from the hospital.

The initial ApoCell dose level (cohort 1) was 35×10⁶ apoptotic cells/kg,on day −1 of transplantation started with recruitment of one patientfirst in order to evaluate preliminary safety profile of ApoCell priorto proceeding with recruitment of additional two subjects to the firstcohort. ApoCell infusion in first patient met the protocol-definedsafety criteria for day 45 and the study proceeded to the second patientwith the same dosage that also met the protocol-defined safety criteria.The study then proceeded to the next phase where an interim analysis ofsafety data was performed by the Data and Safety Monitoring Board (DSMB)after each of cohort 1-3 completed study day 31 and cohort 4 completedstudy day 45. All cohorts met the DSMB criteria and the study wasauthorized to be completed.

The initial plan was to recruit 3 patients for each cohort. However the7th patient received only 90×10⁶ cells/kg instead of 140×10⁶ cells/kgand therefore was immediately included in the second cohort (70×10⁶cells/kg) as an additional patient. In total, 13 patients were treated,three in each cohort one, three, and four, and four patients in cohorttwo.

Eligibility criteria included the following: Adult male or femalesubjects, 18-60 years of age, at the time of screening visit weighing atleast 40 kg and with life expectancy of at least 6 months at the time ofthe baseline visit. Subjects were eligible for allogeneic siblingHLA-matched alloBMT for any disease for which transplantation isappropriate except progressive or poorly controlled malignancies. Theavailability of a genotypically HLA-identical sibling, a phenotypicallyHLA-matched first-degree relative, with at least a 7/8 HLA match at theHLA A, B, C, and DR loci and a clinician decision of a myeloablativeregimen, were required.

Exclusion criteria included: pregnancy, positive serology for HIV,active serious infections, T-cell depleted allograft; Karnofskyperformance status less than 80%, or serious organ dysfunction (e.g.,left ventricular ejection fraction <40%, pulmonary forced vital capacity<60% of predicted, liver transaminases >2.5× the upper limit of normal,or serum bilirubin >3 mg/dL or creatinine >221 μmol/L (2.5 mg/dL).

Conditioning Regimen and Supportive Care

Conditioning regimen for patients were either Busulfan- or Total BodyIrradiation (TBI)-based: for Busulphan: P.O busulphan 16 mg/kg×4 dayswith cytoxan 120 mg/kg or Flu-Bu2-TT2; Fludarabine 30 mg/kg/d for 5 or 6days, I.V. Busulfan, 3.2 mg/kg/d for 2 days or 4 days, I.V. Thiotepa 5mg/kg/d for 2 days (regiment name: FBT). For a TBI-based regimen,Cyclophosphamide 60 mg/kg for 2 days I.V. or etoposide (VP-16) 60 mg/kgwere administered as well as Total Body Irradiation with a TBI dose ofat least 1200 cGy of fractionated TBI. The order of administration ofcyclophosphamide and TBI was at the discretion of the transplant centerwithin each institution, all patients should have received thecyclophosphamide and TBI in the same order. If cyclophosphamide or VP16(etoposide) were given last, there should be at least a one-day restperiod before the peripheral blood stem cell infusion. Fractionated TBIwas administered according to the institutional protocol. Mesna isallowed, but not required. However, each participating center couldemploy conditioning regimen according to their local institutionguidelines provided it was myeloablative and was used for all patientsincluded in the study at that center. Conditioning regimen withanti-thymocyte globulin (ATG) was prohibited (exclusion criteria).

All dosing was based on ideal body weight and ApoCell was givenintravenously on day −1 before transplantation.

GVHD Prophylaxis

Subjects in the study received GVHD prophylaxis regimen that wasprescribed according to the normal standard of care and included IVcyclosporin at a dose of three mg/kg initiated on Day −1 (the dose wereadjusted according to plasma levels) and IV methotrexate at the doses of15 mg/m², 10 mg/m², 10 mg/m² on Days +1, +3 and +6 respectively (threedoses of folinic acid were given starting 18 hours after each dose).Cyclosporin was given orally when the patient was able to swallow andcontinued until Day +90 (in accordance with disease status andchimerism).

Chimerism was assessed by standard cytogenetic analysis in male/femaledonor-recipient. Residual male cells in female chimera were detected byamelogenine gene method. In sex-matched donor-recipient combinations,the VNTR (Variable number of tandem repeats) PCR assay and later on theSTR (short tandem repeats) PCR assay with a 5% sensitivity of detectionwere used to assess the presence of residual host or donor cells.Additional historical controls representing the last decade were takenfrom Gooley et al. (Gooley T A, NEJM 2010).

All infection events were recorded and graded with accordance with theNational Cancer Institute Common Terminology Criteria for AEs (version3). Time to infection was evaluated through Day 180. CMV was tested onscreening, days 3, 10, 17, 31, 45, 66, 100 and 180 study visits.

There were acute follow-up period (days −1 to 3), short-term follow-upperiod (visits on days 10, 17, 31, and 45), and long-term follow-upperiod for acute GVHD (visits on days 66, 100, and 180). The windowvisit were ±2 days for each weekly visit and ±5 days for biweekly ormore visit during the follow up periods.

Regimen-Related Toxicity

Adverse events (AEs) were reported and graded in accordance with CTCAE(version 3). A relationship between the AE and ApoCell versus thosetypically associated with HSCT and GVHD were carefully assigned inaccordance with the guidance in clinical protocol. GVHD severity wasdetermined clinically, however, biopsies of affected organs werestrongly encouraged whenever possible.

Also, the timing of the infusion of the ApoCell product at 24 to 30hours before alloBMT in all cohorts allowed an additional safetyevaluation in this 24 to 30 hour period before the stem cell infusion.

Non-Regimen Related Toxicity and Non-Relapse Mortality (NRM)

Morbidity and mortality related to transplantation included reports ofserious AE (SAE) and documentation of graft failure, veno-occlusivedisease (VOD), sepsis or bacterial infections, noninfectious pneumonia,hemorrhage, refractory GVHD, and multisystem organ failure. Non-fataltoxicity include any SAE or documentation of grade IV ALT, AST, orbilirubin elevation, Grade III serum creatinine, reversible VOD,hemorrhagic cystitis, pericardial effusion, or subdural hematoma.

Diagnosis and Treatment of GVHD

Acute GVHD was graded according to Thomas E D et al. (Thomas E D et al.NEJM 1979) through day 100 of the study. Chronic GVHD (cGHVD) was gradedaccording to Filipovich A H et al. (Biol Blood Marrow Transplant 2005)from day 100 till day 180. The broad category of chronic GVHD includes(1) classic chronic GVHD without features characteristic of acute GVHDand (2) an overlap syndrome in which features of chronic and acute GVHDappear together. In the absence of histologic or clinical signs orsymptoms of chronic GVHD, the persistence, recurrence, or new onset ofcharacteristic skin, GI tract, or liver abnormalities should beclassified as acute GVHD regardless of the time after transplantation.

Engraftment and Donor Chimerism

Neutrophil engraftment was defined as achieving an absolute neutrophilcount (ANC) >0.5×10⁹/L for three consecutive measurements on differentdays. The first of the three days was designated the day of neutrophilengraftment. Platelet engraftment was defined as a platelet count>20×10⁹/L for three consecutive measurements over three or more dayswithout platelet support. The first of the three days was designated theday of platelet engraftment. Subjects must not have had platelettransfusions during the preceding 3 days or in the following 7 daysafter the day of engraftment. The time to a platelet count >100×10⁹/Lwas collected as well. Chimerism was assessed on days 10, 31, 45, 66,100 and 180. Primary graft failure was defined as a lack of neutrophilrecovery in the absence of progressive malignancy affecting the marrow.Secondary graft failure was defined as loss of donor engraftment (<5%donor chimerism) in the absence of progressive malignancy affecting themarrow.

Time to First Hospital Discharge

Time to first hospital discharge was defined as the time from the day ofHSCT (Day 0) to the date of first hospital discharge and was recorded asthe length of the initial hospital stay.

Apo Cell Preparation

The ApoCell product contains apoptotic cells produced from a mononuclearenriched cell fraction from a sibling HLA-matched donor. Eligibilitycriteria for donors included the following: adult male or female donors,18-65 years of age; the donor and recipient must have at least a 7/8 HLAmatch at the HLA A, B, C, and DR loci; above 40 kg; willingness todonate hematopoietic blood mononuclear cells for the generation ofApoCell in addition to the donation for the HSCT. Eligible donorsreturned to the clinic approximately at Day −19 for peripheral bloodmononuclear harvesting using leukapheresis procedure (Cobe® Spectra™,Gambro BCT, Lakewood, Colo., USA) according to the local SOPs. Duringthe approximate 2.5 hours of leukapheresis, 7 L of blood was processedand cells were collected at room temperature into a transfer pack. Theestimated yield of the enriched mononuclear cell fraction from a donorwas 1.0×10¹⁰ cells in an estimated volume of 100-140 ml. The meanpercentage of mononuclear cell fraction in the cell collectionsresulting from the leukapheresis was 88±8% (ranging between 65-96%).Cell yields varied depending on the donor variability. The collectedmononuclear enriched cell fraction from the HLA-matched donors underwentsequential processes for inducing early apoptosis through a multistepprocedure including freezing and thawing the cells followed byincubation with methylprednisolone. The ApoCell final suspensioncontained at least 40% of early apoptotic cells. The cell suspension forinfusion was prepared under current Good Manufacturing Procedures(cGMP). Infusions were performed 24-30 hours before HSCT and within 8hours of completion of preparation. Cells were stored at 2-8° C. untiladministered.

Plasma Biomarkers

Plasma and serum samples were obtained on screening and on study visitdays −1 (before ApoCell infusion), day 0 (before HSCT) and days 3, 10,17, 31, 45, 66, and 100. To ensure optimal recovery, plasma and serumsamples were aliquoted within 2-4 hours after collection and stored at−80° C. until measurement of cytokine levels. Following cytokines weremeasured: TNFR1, IL-2Ra, HGF, IL-8, IL-7, IL-15, IL-6 and IL-1β (forIL-7 measurement highly sensitive kit was used) all from R&D systems(MN, USA). IL-7 and IL-15 were tested from screening till day 31 studyvisits and all other cytokines till day 100 study visit unless statedotherwise. ELISAs were performed in duplicates. Plates were analyzedwith Infinite F50 absorbance reader (Techan, Austria) using Magellansoftware. The results are presented as median concentration levels.

Statistical Analysis

Descriptive statistics were used to summarize outcome measures andbaseline characteristics. In this analysis all available data werepresented with no imputation for any missing data. Subjects contributedthe data available up to the point of withdrawal or study completion ordeath. Descriptive statistics including means, median, standarddeviation (SD), minimum and maximum values were used to summarizecontinuous variables. Dichotomous variables were presented as count andpercentages. Student-t-test was used to compare mortality and GVHDoccurrence to historical controls and previous reports. All subjects whoreceived the ApoCell infusion were included in the safety analysis.Student t test (two tail type 1) was used for potency assay analysis.

Historical Controls

Historical control patients were selected from the computerized registryat the Bone Marrow Transplantation & Cancer Immunotherapy Center,Hadassah University Hospital according to the following rules: underwentallogeneic stem cell transplantation from a matched sibling donor andhave similar age, sex, disease, disease status and conditioning regimento the current study patients. The data was verified from the patients'electronic files prior to analysis. Control group consisted of 25patients (18 were males and 7 females), with median age of 26 years(range 9-63). All patients were referred to the Hadassah Hospital forBMT between 1982 and 2009. As it was a prerequisite for the patients'selection for this analysis, all patients were transplanted from fullymatched HLA class I and II family members (24 siblings and 1 father).

Engraftment data was found for 21 patients. Late rejection occurred inone patient (5%). Engraftment of neutrophils was achieved in all (21 outof 21) patients at a median of 14 days (range 9-22 days). Engraftment ofplatelets was achieved in 16 out of 18 evaluable patients at a median of12.5 days (range 9-32 days).

Eighteen of the 25 (72%) control patients were discharged from theinitial transplant admission. The median hospital stay was 40.5 days(range 27-79 days).

GVHD: Fifteen of the 25 control patients (60%), developed acute GVHDgrade I-IV at a median time of 26 days. The incidence of grade II-IVacute GVHD was 50% (12 of 24 patients and III-IV acute GVHD was 20%(5/25 patients).

Transplant-related mortality (TRM): 7 out of 25 (28%) patients diedduring the first 100 days due to transplant related complications. Noneof the surviving patients died between days 100 to 200 from transplantcomplications, thus TRM was kept at 28% at 200 days. Death was causeddue to infection in 3 patients, GVHD in one, GI bleeding in one,rejection in one and cardiac arrest in one.

Transplant-related toxicity (TRT): creatinine—4 of the 18 patients (22%)with available kidney function data had renal failure defined ascreatinine >1.5× upper normal levels up to day +200. In 2 of them renalfailure was severe and necessitated intervention. Bilirubin—7 of the 18patients (39%) with available bilirubin level data experiencedsignificant hepatotoxicity defined as bilirubin >2× upper normal levelsup to day +200.

The following methods were employed in Examples 5-10 disclosed herein.

Cell Cultures and Reagents.

Cells were cultured in Dulbecco's modified Eagle's medium (DMEM), withhigh glucose supplementation (Invitrogen-Gibco, Carlsbad, Calif.), andwith 1% L-glutamine (Biological Industries, Israel), 10% fetal bovineserum (Biological Industries), and 10 μg/ml ciprofloxacin (SigmaAldrich, Israel). The caspase-1 inhibitor z-YVAD-fmk, nigericin, andbafilomycin A1 were purchased from Calbiochem (Darmstadt, Germany)N-acetyl-L-cysteine (NAC) and lipopolysaccharide (LPS) were from SigmaAldrich. DSS reagent was from MP Biomedicals (Illkirch, France). Forimmunostaining, the following antibodies were used: anti-COX2 (CaymanChemicals, Ann Arbor Mich., USA), anti-myeloperoxidase (ThermoScientific, Waltham Mass., USA), anti-phospho-IκBα andanti-phospho-NF-κB p65 (Cell Signaling, Danvers Mass., USA).

Generation of Apoptotic Cells.

A composition containing human apoptotic cells (ApoCell) was producedfrom the mononuclear enriched cell fraction of healthy volunteer donorsin a leukapheresis procedure. During leukapheresis, approximately 300 mlof autologous plasma was collected from the donor for subsequent use inthe preparation of the apoptotic cells. The plasma was collected in atransfer pack, aliquated and frozen for 2 hours at −80° C. and thenstored at −18-(−25°) C. till manufacturing. Collected cells met allspecifications for harvested cells as described in Table 3 above.Differential count was performed on the SYSMEX hematology analyzer. Cellviability was determined by propidium iodide staining by flow cytometry.Following collection, cells were washed with RPMI 1640 supplemented with2 mM L-glutamine, 10 mM Hepes and 5% of ACD formula A containing 10 U/mlHeparin and frozen at 5-6.5×10⁷ cells/ml in freezing bags. The finalformulation of the freezing medium was RPMI 1640 supplemented with 2 mML-glutamine, 10 mM Hepes, 5% Hes, and 10% dimethyl sulfoxide (DMSO)containing 20% autologous plasma and 5% of ACD formula A containing 10U/ml Heparin. Next, cells were thawed and washed with RPMI 1640supplemented with 2 mM L-glutamine, 10 mM Hepes and 5% of ACD formula Acontaining 10 U/ml Heparin. Cells were then re-suspended to a finalconcentration of 5×10⁶/ml in RPMI 1640 supplemented with 10 mM Hepes, 2mM L-glutamine, with addition of 10% autologous plasma, 5% of ACDformula A containing 10 U/ml Heparin and 50 μg/ml methylprednisolone andincubated for six hours in LifeCell flasks at 37° C., 5% CO₂. Followingincubation, cells were harvested, washed with PBS and re-suspended atthe desired concentration in PBS.

Apoptotic Cell Measurement.

Apoptosis was assessed using Annexin V and propidium iodide (PI)apoptosis detection kit (MBL International, Woburn Mass., USA). Cellswere acquired with a FACSCalibur instrument and analyzed using FCSExpress software (De Novo, Los Angeles, Calif., USA). Apoptotic cellsroutinely contained at least 40% AnnexinV and <5% PI-positive cells wereused in all experiments.

Isolation of peritoneal macrophages. Primary resident peritonealmacrophages (pMΦ) of WT or Nlrp3−/− mice were generated as describedelsewhere (Bauer et al. 2010). Briefly, mice were sacrificed underisoflurane anesthesia by cervical dislocation. Peritoneal lavage wasthen performed by exposing the parietal peritoneum and injectingintraperitoneally with a transpipette 10 ml of 2% FBS in PBS. Theperitoneal lavage fluid was centrifuged and re-suspended at the desiredconcentration. The adherent pMΦ (F4/80 positive cells) subset consistsabout 20% of the peritoneal lavage as observed by flow cytometeranalysis. Cells were then plated into culture dishes overnight. Cellswere washed, and adherent cells were used for cytokine assays. Whereindicated, experiments were performed after four weeks of co-housing WTand NLRP3-deficient mice to neutralize the microbiota effect.

IL-1β ELISA.

pMΦ were seeded into 96-well plates at a density of 2×10⁵ cells perwell. After LPS priming for 1 h, cells were stimulated with differentactivators for 24 h. Cell culture supernatant was used for ELISA(Biolegend, San Diego Calif., USA), which was performed according to themanufacturer's protocol.

Western Blotting.

The processed IL-1β p17 subunit and activated caspase-1 p10 subunit andtheir release into the culture supernatant were determined by Westernblotting. In brief, 12 hours after indicated activators addition, thesupernatant was collected and suspended in SDS-PAGE sample buffer, andheated to 85° C. for 10 min Macrophages were lysed in lysis buffer (50mM Tris-HCl pH8.0, 5 mM EDTA, 150 mM NaCl, 1% Triton-X 100 and aprotease inhibitor cocktail (Roche)) and stored at −80° C. untilanalyzed. Protein from 1×106 macrophages was loaded per well of a 15%acrylamide gel and transferred to a PVDF (poly(vinylidene difluoride))membrane by electroblotting. Western blots were performed withanti-mouse IL-1β antibody (clone B122; Biolegend) diluted 1:500 andanti-mouse caspase-1 p10 antibody (Santa Cruz) diluted 1:1000.Appropriate HRP-conjugated secondary antibodies (Jackson ImmunoResearchLaboratories, West Grove, Pa., USA) were used and proteins detectedusing ECL reagent (Biological Industries). An anti-mouse actin served asa loading control.

Induction of Colitis.

Colitis was induced by oral administration of 3% (w/v) DSS solution(m.w. 36,000-50,000; MP Biomedicals) ad libitum in drinking water for7-9 days until sacrifice. The control group received distilled water (0%DSS) during the same time. Where apoptotic cell treatment was applied,mice received a single infusion into tail vain containing 25-30×106cells/150 μl in PBS. Control mice received 150 μl PBS.

Induction of Colitis by Adoptive T-Cell Transfer.

Naïve CD4⁺CD45RB^(high) T cells were isolated from spleens of C57BL/6mice via FACS sorting as previously described (Izcue, 2008). In brief,after negative enriching for CD4+ lymphocytes, single-cell suspensionswere stained with APC-conjugated anti-CD4 and FITC-anti-CD45RB (allobtained from biolegend). Naïve CD4⁺CD45RB^(high) T cells were purified(>99%) with a FACSAria cell sorter (BD Biosciences, San Jose, Calif.).The CD4⁺ CD45RB^(low) population was also sorted and served as thenegative control. Sex-matched Rag1^(−/−) recipient mice received 5×10⁵CD4⁺CD45RB^(high) or CD4⁺CD45RB^(low) T cells by intraperitoneal (i.p.)injection, and development of intestinal inflammation was monitored asdescribed below. Groups receiving apoptotic cell treatment were injectedwith a dose of 30×10⁶ ApoCell/150 μl PBS per mouse on indicated days viatail vain. Control groups were injected with 150 μl PBS only.

General Assessment of Colitis.

Mice were sacrificed when symptoms of clinical disease became apparentin control groups, usually around 7-9 days in DSS-model and 8 weeks inTCT model. IBD was assessed using a standard IBD Clinical Score by dailymeasurements of weight change, stool consistency, and hematochezia, asdescribed elsewhere (Hartmann, 2000), with modification. No weight losswas counted as 0, weight loss of 1 to 5% as 1, 5 to 10% as 2, 10 to 20%as 3, and >20% as 4 points. For stool consistency, 0 points were awardedfor well-formed pellets, 2 for pasty and semi-formed stools that did notstick to the anus, and 4 for liquid stools that did stick to the anus.Bleeding was scored as 0 points for no blood in hemoccult, 2 forpositive hemoccult, and 4 for gross bleeding. These scores were added toform a total clinical score that ranged from 0 (healthy) to 12 (maximalcolitis activity). After sacrificing the animals, colons were dissectedand fixated in 4% formaldehyde, and embedded in paraffin before stainingwith hematoxylin and eosin. Histological quantification of mucosaldamage, presence and extent of inflammation, crypt damage, and percentinvolvement, with a range from 0 to 4, was performed on distal colonsections of the specimens. Specimens and treatment groups were blindedbefore histological quantification.

Measurement of Reactive Oxygen Species (ROS).

Production of ROS by the inflammasome triggering agent DSS was measuredwith the ROS detection kit (Enzo Life Sciences, Farmingdale N.Y., USA).pMΦ from female B6 mice were seeded onto eight-chamber slides at density0.1×106 cells/chamber, and cultured at 37° C. overnight. Thereafter, pMΦwere washed twice with PBS, treated for two hours with apoptotic cellsat a 1:8 ratio, washed, primed with LPS, and treated with 3% DSS for anadditional 30 minutes. Negative control cells were treated with mediaonly. After washing, the cells were suspended in 200 μl of DMEM andstained with the ROS detection reagent (1 μM) for 30 minutes.DSS-induced intracellular ROS was detected by fluorescence microscopeexamination at 488 nm excitation wavelength with a 525-nm emissionfilter. (Original magnification ×100). Where flow cytometer detectionwas applied, MΦ were detached by trypsin-EDTA after treatment, washed,and analyzed using an LSRII instrument (BD Biosciences).

Lysosomal Stability Evaluation.

Lysosomal damage by DSS challenge was evaluated by acridine orange stainas described elsewhere (Bauer et at 2010). Briefly, peritonealmacrophages were plated into 24-well culture dishes overnight, whichafter non-adherent cells were washed with PBS. The remaining adherentmacrophage cells were introduced to apoptotic cells (1:8) for two hours.Macrophages were then washed, primed with LPS during one hour andstimulated for 24 h with DSS. Cells were then washed and incubated with0.25 μg/ml acridine orange for 15 minutes for lysosome stain. Lysosomaldamage was determined as loss of fluorescence intensity emission at600-650 nm with an LSRII (BD Biosciences).

Immunohistochemistry.

Paraffin-embedded slides from Balb/c mice were deparaffinized andincubated in 3% H2O2. Antigen unmasking was carried out by microwaveheating (20 min) in 10 mM Tris buffer containing 1 mM EDTA. Slides wereincubated with primary antibodies anti-COX2, anti-MPO, anti-pNF-κB, andanti-pIκBα diluted in CAS-Block (Invitrogen), or with CAS-Block alone asa control. Appropriate secondary antibodies (Nichirei) were then addedand slides were incubated at room temperature for 30 min Color wasdeveloped using the DAB substrate kit (Thermo Scientific) followed bycounterstaining with Mayer's hematoxylin (Sigma Aldrich). Controlswithout addition of primary antibody showed low or no backgroundstaining in all cases.

Animals and Co-Housing.

BALB/c or C57BL/6 mice were obtained from Harlan Inc. (Jerusalem,Israel). Mice were all female and 8-10 weeks of age upon arrival. Whereindicated, experiments were performed after 4 weeks of co-housing WT andNLRP3 deficient mice to neutralize the microbiota effect.

Statistical Analysis.

All data are expressed as mean±SEM. The statistical significance of thedifferences was evaluated by unpaired t-test (two-tailed, except whereindicated otherwise) or one way ANOVA with Tukey's multiple comparisontests. P values of 0.05 or less were considered to be statisticallysignificant.

Results Example 1 Infusion of Allogeneic Apoptotic Cells as Prophylaxisof GVHD in Myeloablative Allogeneic Bone Marrow Transplantation is Safe

Patient, Donor, and Graft Characteristics

Median total number of cell transplanted and CD34+ cells infused intorecipients were 13.6×10⁸/kg (range, 9.3-29.5 10⁸/kg) and 7.2×10⁶/kg(range, 3.7-22.4×10⁶/kg), respectively (Table 1). Patient, disease, andtransplantation characteristics are summarized in Table 1. The mostcommon diagnosis was acute lymphoblastic leukemia (ALL; n=7, 54%),followed by acute myeloid leukemia (AML; n=5, 38%) and one patient withchronic myeloid leukemia (CML; n=1, 7.7%). In patients with AML, onepatient presented with disease de novo, and four patients developed AMLfrom an antecedent myelodiplastic syndrome (MDS). A total of fivepatients with ALL were in first complete remission (CR1), one patientwas in second complete remission (CR2) and one patient in second partialremission (PR2). A total of five patients with AML were in CR1. Thesingle patient with CML was in chronic phase and was unresponsive tothree tyrosine kinase inhibitor (TKI) before transplantation. Allpatients received related donor allografts. The median patient age was37 years (range, 20-59). In addition to HLA match on HLA A, B, C (wasevaluated in 8 of 13 patients) and DR loci, DQ was also evaluated in 12of 13 patients. HLA-matching data is presented at Table 1.

TABLE 1 Patient and BMT characteristics Infused cell doses ConditioningDisease Total (×10⁸/kg)/ regimen severity Patient CD34+(×10⁶/kg)myeloablative HLA-matching Diagnosis Weight Gender Age Cohort 12.5 ×10⁸/kg/ Flud Busflex CR1; L2 ALL 81 M 38 Cohort 3.69 × 10⁶/kg Thiotepa1-1 Cytarasine 20.34 × 10⁸/kg/ Cytoxan PR2; L1 ALL 64 F 38 Cohort 22.38× 10⁶/kg TBI, ARAC 1-2 and Methotrexate 29.5 × 10⁸/kg/ Cytoxan, TBI CR1;T-ALL ALL 58 M 30 Cohort 7 × 10⁶/kg 1-3 10 × 10⁸/kg/ Cytoxan, TBI CR2;L2 ALL 77 M 25 Cohort 6.8 × 10⁶/kg 2-1 13.6 × 10⁸/kg/ Flud Busflex CR1MDS, 83 F 59 Cohort 15.8 × 10⁶/kg Thiotepa AML 2-2 24.8 × 10⁸/kg/Cytoxan, TBI CR1; L1 ALL 40 M 24 Cohort 8.1 × 10⁶/kg 2-3 9.3 × 10⁸/kg/Flud Busflex CR1; M0 MDS, 78 M 58 Cohort 18.6 × 10⁶/kg Thiotepa AML 2-418 × 10⁸/kg/ Busulphan CR1 MDS 100 M 49 Cohort 7.2 × 10⁶/kg Cytoxan AML3-1 21.6 × 10⁸/kg Busulphan CR1; M1- MDS, 96 M 37 Cohort 9.7 × 10⁶/kgCytoxan M2 AML 3-2 19.3 × 10⁸/kg/ Cytoxan CR1; T- ALL 85 M 23 Cohort16.6 × 10⁶/kg TBI ALL 3-3 10.2 × 10⁸/kg/ Cytoxan TBI CR1; L2 ALL 72 M 20Cohort 6.35 × 10⁶/kg 4-1 10.5 × 10⁸/kg/ Busulphan Chronic CML 51 F 37Cohort 7.19 × 10⁶/kg Cytoxan phase 4-2 11 × 10⁸/kg/ Flud Busflex CR1; M2AML de 111 M 40 Cohort 6.55 × 10⁶/kg novo 4-3Potency Assay

Tolerogenic DC can be generated upon interaction with apoptotic cells(Verbovetski I J Exp Med 2002) or apoptotic cell products (Krispin ABlood 2006). To each ApoCell preparation, the tolerogenic effect of theprepared ApoCell was specifically examined using in vitro interactionwith immature dendritic cells (iDCs). iDCs express low level of HLA-DRand co-stimulatory molecules. Following the exposure to maturationstimuli like LPS, iDCs undergo maturation and upregulate expressionlevels of HLA-DR and costimulatory molecule CD86.

Potency assay results from 13 ApoCell preparations infused to thepatients are summarized in Table 2. The results represent averagepercentage of inhibition in maturation of LPS treated DCs (inhibition inDR and CD86 expression) following interaction with ApoCell. As shown inTable 2, significant and dose-dependent down regulation was seen.Representative results from one patient potency assay are shown in FIG.1.

TABLE 2 Potency assays summary from 13 ApoCell preparations % Inhibitionof DCs:ApoCell P-Value maturation by LPS ± STD ratio Marker 0.0146 30.4± 25.8 1:2 DR 0.0002 52.1 ± 35.7 1:4 0.0002 67.2 ± 31.8 1:8 0.0147 40.5± 25.7 1:2 CD86 0.0004 66.3 ± 26.2 1:4 0.0000 81.0 ± 20.5 1:8Engraftment

The median time to neutrophil recovery for recipients was 13 days(range, 11-19), and the median time to platelet recovery was 15 days(range, 11-59). Median time to neutrophil and platelets engraftment inthe first cohort was 13 days (range 13-14 days) and 17 days (range11-59) respectively; in the second cohort, 14 (range 11-17) and 14(range 11-18), respectively; in the third cohort, 14 days (range 12-19)and 15 days (range 13-54) respectively; and in the fourth cohort 12 days(range 11-13) and 15 days (range 13-17) respectively.

Ten of 12 patients (83%) with available data of chimerism on day 31 ofthe study were donor type. One additional patient had poor technicaltest that yielded no result on day 31 and was found to be donor type onthe following visit (day 45). 100% of patients converted to donor typeby day 66. Primary graft failure did not occur. None of the patientswith mixed chimerism at day 31, were found to have early diseaserelapse.

Adverse Events

Ten SAEs were reported with all being not related (seven) or unlikely tobe related (three) to ApoCell infusion. Documented SAEs were: two septicshocks, two relapses, one hemorrhagic cystitis, one gastroenteritis dueto adenovirus infection, one vomiting, and three incidences of fever.Out of hundreds AE, only three were reported as possibly related toApoCell infusion (with no definite or probable AE related to ApoCell);one hypotension on the day of the infusion (day −1), one throatirritation on the day of the infusion (day −1) and one relapse on day131 of the study.

Relapse

The cumulative incidence of relapse at 100 days and 180 days aftertransplantation was 7.7% (n=1) and 31% (n=4), respectively. Three offour patients with relapse (75%) had ALL. All received cyclosporine.

Survival

Overall survival on day 45, 100 and 180, was 100%, 92.3% and 84.6%,respectively (FIG. 2A). Survival not related to relapse was 100% on day45, 92.3% on day 100 and on day 180 (FIG. 2B). Transplantation relatedmortality (TRM) was 0% on day 45, 7.7% on day 100 and 7.7% on day 180.Only one patient in the treatment group (7.7%) died (FIG. 2C; column 1),compared to 7 (28%) of the matched historical controls from hospitalrecords (FIG. 2C; column 2) and 16% in the retrospective survey (datanot shown).

Time to First Hospital Discharge

Mean time to first hospital discharge for all 13 patients was 34.2 days(range 15-103 days). Mean time to first hospital discharge for threepatients treated in the first cohort dose (35×10⁶/kg) was 46.3 days(range 15-103 days); mean time to first hospital discharge for fourpatients treated in the second cohort dose (70×10⁶/kg) was 33.5 days(range 20-87 days); mean time to first hospital discharge for threepatients treated in the third cohort dose (140×10⁶/kg) was 24.3 days(range 22-28 days) and mean time to first hospital discharge for 3patients treated in the last cohort dose (210×10⁶/kg) was 18.3 days(range 17-21 days) (FIG. 3).

The results presented here suggest that single-infusion of donor earlyapoptotic cells (ApoCell) as prophylaxis of graft-versus-host disease inmyeloablative allogeneic bone marrow transplantation is safe. ApoCellwas given 24 hours before BMT and no related or possibly related SAEsspecific for ApoCell infusion were reported. A total of ten SAE werereported with all being or not related (seven) or unlikely to be related(three) to ApoCell infusion. Out of hundreds AE, only three werereported as possibly related to ApoCell infusion: hypotension on the dayof the infusion, throat irritation also on the day of the infusion, andrelapse on day 131 of the study. No definite or probable adverse effectsrelated to ApoCell were reported. In addition, no prolonged time toengraftment, duration of hospitalization, chimerism delay, increasedrate of mortality, CMV or any serious infections, and relapses, wasobserved when compared to historical controls and similar patientsdescribed in the literature (Gooley ibid.), in all doses examined.

Example 2 Infusion of Allogeneic Apoptotic Cells Reduces High Grade GVHDin Patients Undergoing Myeloablative Allogeneic Bone MarrowTransplantation

Acute GVHD was assessed through day 100 of the study in 12 out of 13patients and in one patient in the second treatment group through day87. All were included in the day-100 cumulative incidence. The day-100cumulative incidences of grades II through IV and III through IV acuteGVHD (aGVHD) for all patients were 23.1% and 15.4%, respectively (FIG.4). Acute GVHD was assessed through day 180 of the study in 10 out of 11patients. The median times to onset of grades II through IV and IIIthrough IV acute GVHD were 31 days (range, 31-44 days) and 47 days(range, 31-62 days), respectively. No patient developed acute GVHDbeyond day 100 after transplantation. Ten of 11 patients were assessedfor aGVHD at day 180. One of 10 patients (10%) was with persistent skinaGVHD grade 1 with overall severity grade 1 on day 180 of the study. Ofnote is that no high grade GVHD (grade II-IV) was documented in twocohorts treated with the highest dose of ApoCell (FIG. 4A). GVHD gradeII-IV incidence in transplanted patients receiving single infusion ofapoptotic cell preparation was significantly low (7.7% of 13 subjects,FIG. 4B; column 1) in comparison to historical control from hospitalrecords (50% of 25 subjects, FIG. 4B; column 2) and reports from theliterature (71%, data not shown; Gooley, ibid.)

In every successful treatment that potentially avoids high grade aGVHD,there arises a question regarding the possible loss ofgraft-versus-leukemia (GVL) effect that was found to correlate to theseverity of GVHD (Horowitz M M Blood 1990). In the present study therelapse rate was 7.7% at 100 days and 30.8% at 180 days. The relapserate at 100 days is not different from the reported relapse rate ofsimilar patients undergoing alloBMT. The rate of 30% relapse by day 180may be considered borderline high, however, 75% of relapse incidenceswere patients with ALL (3 of 4 patients that tend to relapse in highrates of 70% at similar age group). Furthermore, although, the gradeII-IV GVHD was decreased to 0 in the two higher doses, grade I aGVHD wasincreased to 50% in the same cohorts, indicating that ApoCell treatment,as a physiological modality, reduces high grade GVHD rather thanabolishing it.

Chronic GVHD (cGVHD) was assessed from day 100 till day 180 of thestudy. Ten of 11 patients were assessed for cGVHD at day 180. Five of 10patients (50%) had mild cGVHD involving skin (four patients) andconjunctiva (one patient).

Example 2 demonstrates that the incidences of acute grades II through IVand grades III through IV GVHD were very low (23% and 15% respectively)in comparison to historical control and reports from the literature(Gooley, ibid.) 71% grade II-IV and 14% grade III-IV in last decade).Remarkably, in the two higher dosages there was 0% aGVHD grade II-IV,suggesting a remarkably effective prophylactic treatment.

Example 3 Reduced Incidence of Hepatotoxicity in Transplant PatientsReceiving a Single Dose of Apoptotic Cells

The number of transplant patients developing hepatotoxicity in all fourof cohorts I-IV (n=13; column 2), receiving from 35-210×10⁶ apoptoticcells was compared with that of matched controls from hospital records(n=18), and with the long-term documented transplant patients (n=1148;Gooley et al, ibid.).

Only one patient in the treatment group developed hepatotoxicity (7.7%;FIG. 5A, column 2), compared to 39% (FIG. 5A, column 1) of the matchedhistorical controls and 20% (FIG. 5A, column 3) in the long-termdocumented transplant patients. Of note, no hepatic toxicity of GVHD wasdocumented in the three higher dosages treatment groups (cohorts II-IV)treated with the apoptotic cell preparation of the invention (FIG. 5B),compared to 39% observed among the matched historical controls.

Example 4 Validation of the Clinical Studies Using Acute GVHD (aGVHD)Plasma Biomarkers

Plasma biomarkers that were reported as aGVHD discriminators or aGVHDpredictors were examined in order to further validate the clinicalresults. A panel of four plasma biomarkers IL-2Ra, TNFR1, IL-8 and HGFwere proposed as markers that can help optimally discriminate patientswith and without aGVHD and can confirm the diagnosis of GVHD in patientsat the onset of clinical symptoms of GVHD and can also provideprognostic information for survival independent of GVHD severity.

First, plasma levels of TNFR1, IL-2Ra, IL-8 and serum levels of HGF wereevaluated. As shown in FIGS. 6A,B,E and F, clearly higher levels werefound in aGVHD grade II-IV group in comparison to aGVHD grade 0-I group.Median TNFRI level in aGVHD grade 0-I group at day +10, +17, and +31were 2172 pg/ml, 2530 pg/ml, and 2698 pg/ml, respectively.

However, in aGVHD grade II-IV group, TNFRI median levels were 3171pg/ml, 3301 pg/ml and 4342 pg/ml respectively. Similarly, IL-2Ra medianlevels in aGVHD grade 0-I group were 3650 pg/ml, 2916 pg/ml, and 2455pg/ml, respectively, in comparison to 4601 pg/ml, 8102 pg/ml and 3624pg/ml in aGVHD grade II-IV group, respectively. Once again, HGF medianlevels in aGVHD 0-I group at day +17, +31, and +45 were 1517 pg/ml, 1464pg/ml, and 1873 pg/ml, respectively in comparison to 2418 pg/ml, 3264pg/ml and 2326 pg/ml in aGVHD grade II-IV group, respectively. Finally,median levels of IL-8 in aGVHD grade 0-I group at day +10, day 17 and+31 were 48.3 pg/ml, 22.3 pg/ml and 37.1 pg/ml, respectively, incomparison to 90.8 pg/ml, 41.9 pg/ml and 61.8 pg/ml in aGVHD grade II-IVgroup, respectively (FIGS. 6 A,B,E and F).

As some publications prefer using median concentration ratio rather thancytokine concentration, especially with TNFRI levels (Ferrara J L Bestpractice & research clinical haematology 2007, Choi S W et alTransplantation 2012). However, when the inventors used TNFRI ratio orother cytokine ratios our results did not change (data not shown).

Two additional cytokines, IL-15 and IL-7 were reported to correlate toaGVHD. As shown in FIG. 6C, IL-15 median plasma levels in our study weresignificantly higher in aGVHD grade II-IV group 33.5 pg/ml, 30 pg/ml, a42 pg/ml and 12 pg/ml, at days 0, +3, +10, +17, respectively, incomparison to aGVHD grade 0-I group; 18.6 pg/ml, 15.7 pg/ml, 15.5 pg/ml,and 7.0 pg/ml, respectively.

IL-7 was also elevated in high grade GVHD (FIG. 6G), although the mainelevation was seen in grade III-IV with 8.9 pg/ml, 32.6 pg/ml and 23.0pg/ml at days +10, +17 and +31, respectively, versus, 4.5/ml, 10.5 pg/mland 9.0 pg/ml, respectively in aGVHD 0-I.

As cytokine control, IL-6 and IL-1b were measured. As expected, othercytokines showed elevations that did not distinguish between GVHDgrades.

Example 4 demonstrates plasma biomarkers supporting the clinical datapresented herein. Although published data is lacking regarding what arethe best biomarkers, plasma levels of six different biomarkers: TNFRI,IL-2Ra, HGF, IL-8, IL-15 and IL-7, distinguished well between high tolow grade or no-GVHD. Additional two control cytokines (IL-1b and IL-6)further emphasized the specificity of findings.

Example 5 The Ameliorative Effect of the Apoptotic Cell Composition onInflammatory Colitis

The therapeutic effect of a single infusion of the apoptotic cells inameliorating colitis was examined in two IBD models: adoptive T celltransfer (TCT) of naïve CD4 cells and dextran sulfate sodium(DSS)-induced colitis.

First, CD45RB^(high) Naïve T cells were harvested and sorted from WTC57BL/6 mice and adoptive transferred into immune deficient mice lackingthe RAG enzyme. Non naïve (CD45RB^(low)) T cells were used as a controlfor transferred cells. A composition comprising early apoptotic cellswithin 150 μl of PBS was administered to the mice on the day of theT-cell transfer, while 150 μl of PBS were used as a control.

Four weeks later, mice in the PBS treated group started to developclinical signs of IBD, namely weight loss and pasty stool discharges.However, in the group treated with apoptotic cell composition, noclinical deterioration was observed. The CD45RB^(high) mice group lost17% of their initial body weight whereas the apoptotic cell treatedgroup gained weight (FIG. 7A, p<0.03). Similarly, the overall clinicalscore was significantly reduced in the group treated with apoptoticcells compared to non-treated group (FIG. 7B, p<0.02). Elevation inmesenteric T-regulatory cells in the treated group was also demonstratedin mesenteric lymph nodes (FIG. 7C).

Example 6 DSS Induces Caspase-1-Dependent Pro-IL-1β Processing Via NLRP3Inflammasome

Enhanced production of IL-1β has been previously shown to be detectedupon exposure of murine macrophages to DSS, and more recently was shownin vitro and in vivo to be NLRP3 inflammasome-dependent.

In order to investigate the possible role of apoptotic cells in negativeregulation of the inflammasome, murine macrophages were generated andexposed to DSS. In agreement with previous observations, DSS was foundto induce IL-1β release from murine macrophages, as can be seen in FIG.8. A combination of Toll Like Receptor (TLR) triggering with LPS andinflammasome triggering with DSS led to a marked IL-1β secretion. As canbe seen in FIG. 8, inhibition of caspase-1 by the specific inhibitorz-YVAD-fmk peptide led to an almost complete inhibition of IL-1β release(p<0.05, unpaired t test), demonstrating the role of caspase-1 inDSS-mediated IL-1β release. Activation of the NLRP3 inflammasome is K⁺efflux-dependent, lysosomal-dependent, and ROS-dependent. Indeed,blocking K⁺ efflux with high concentrations of KCl inhibitedDSS-mediated IL-1β release (FIG. 9A, p<0.05, unpaired t test) Similarly,blocking lysosomal acidification with bafilomycin, an inhibitor ofvacuolar ATPase, inhibited IL-1β secretion (FIG. 9B, p=0.05, unpaired ttest). Finally, inhibition of ROS generation by N-acetyl-L-cysteine(NAC) significantly inhibited IL-1β secretion (FIG. 9C, p<0.05, unpairedt test).

To further support the finding that DSS-mediated IL-1β secretion iscaspase-1 and NLRP3 activation-dependent, wild-type and NLRP3-deficientmice were co-housed and macrophages were extracted from the mice andexposed to DSS. As can be seen in FIG. 9D, DSS-induced IL-1β secretionwas significantly reduced in macrophages extracted from NLRP3-deficientmice (p<0.02, t test).

Example 7 Treatment with the Apoptotic Cell Composition Shows anAnti-Inflammatory Effect in a Dextran-Sulfate-Sodium (DSS) Model

The effect of treatment using the apoptotic cell composition was furtherexamined in a DSS-mediated intestinal inflammation model in vivo byadministering 3% DSS for a period of 8-9 days to Balb/c mice withintheir drinking water Inflammatory bowel disease (IBD) score parameters,including body weight, the presence of latent or gross blood per rectum,and stool consistency, were determined daily.

Mice treated with apoptotic cells in addition to DSS showedsignificantly less body weight loss starting from day 6, as compared tomice treated only with DSS (FIG. 10A, p<0.05 and 0.001, t test).Clinical score analysis revealed significantly less severe colitis inmice treated with apoptotic cells, in all parameters evaluated (FIG.10B, p<0.01, t test). On macroscopic examination, DSS-treated colonswere severely inflamed and hyperemic, and contained less feces due tomassive diarrhea. When treated with apoptotic cells, colons were lessaffected and were longer than colons of mice treated only with DSS(9.4±0.14 cm vs. 8.9±0.2, p<0.05) (FIG. 10C).

Next, IL-1β levels were measured in colonic homogenates of mice treatedwith DSS, either with or without treatment with the apoptotic cellcomposition. After 7 days of DSS intake, IL-1β levels were significantlyelevated in colons of mice not treated with the apoptotic cellcomposition (FIG. 10D, p<0.02, t test). However, in mice treated with asingle apoptotic cell injection prior to DSS intake, a similar elevationwas not observed.

In order to further establish the exhibited observations, histologicaland immunohistochemical analysis of colonic tissue were obtained on day9 following DSS intake. Biopsies showed significantly less severemucosal infiltration by inflammatory cells and reduced tissue damage inmice treated with apoptotic cells, translating into a significantlyimproved histological colitis severity score (FIG. 11A I-III, p<0.05, ttest). This analysis was performed by a pathologist blinded to thedifferent groups. Since IL-1β is known to induce accumulation ofneutrophils at inflammation sites, the range of neutrophils in coloninflammation was evaluated. Neutrophil infiltration was markedly higherin colon tissue of mice which did not receive apoptotic cell treatment.To further illustrate the dramatic reduction in neutrophil infiltrationwith apoptotic cell treatment, myeloperoxidase (MPO) staining wascombined with hematoxylin staining (FIG. 11B). Indeed, DSS-treated miceshowed a dramatic increase in MPO-stained neutrophils in the colon,while a single treatment with apoptotic cells markedly reducedMPO-stained neutrophil accumulation.

Cyclooxygenases (COXs) catalyze a key step in the formation ofpro-inflammatory prostaglandins and have been shown to be induced byIL-1β. The main product of the Cox2 cascade is PGE2, which is the keymediator in the acute inflammatory response. Indeed, Cox2 immunostainingshowed a dramatic elevation in the number of positive cells inDSS-treated colons compared to non-treated colons (FIG. 12). Whenapoptotic cell treatment was applied, a marked reduction was observed.

Example 8 In Vivo NF-κB Inhibition by Apoptotic Cells in DSS-InducedColitis

NF-κB is normally sequestered in the cytoplasm by means of associationwith an inhibitory protein, IκBα. Activation of NF-κB involvesstimulation of the IKK complex, which phosphorylates IκBα, triggeringits degradation and the nuclear translocation of active NF-κB. Toexamine NF-κB signaling, the phosphorylation of IκBα in colonic tissuefrom mice with DSS-induced colitis was examined and compared to miceexposed to DSS following treatment with apoptotic cells. An appreciablyhigher number of pIκBα-positive cells were observed in colon treatedsolely with DSS compared with colon that was also treated with apoptoticcells (FIG. 13). Inhibition of NF-κB signaling was further confirmed bythe reduced number of cells that were positive for nuclear phospho-p65NF-κB, detected by immunostaining (FIG. 14).

Example 9 Apoptotic Cells Inhibit Inflammasome-Induced IL-1β Releasefrom Macrophages

While inhibition of IL-1β release by macrophages exposed to TLR agonistshas been demonstrated, it is not known whether they can inhibitsecretion upon NLRP3-specific activation. In order to examine the effectof the apoptotic cell composition on inflammasome-induced IL-1β release,isolated macrophages were exposed to lipopolysaccharide (LPS) and DSSwith or without earlier interaction with apoptotic cells for two hours.

Incubating apoptotic cells with macrophages had no effect on IL-1βsecretion in the absence of TLR and inflammasome triggering. However,prior apoptotic cell treatment in the presence of LPS and DSS,significantly inhibited IL-1β secretion from macrophages (FIG. 15A,p<0.01, unpaired t test), with similar inhibition rates as of z-YVAD,KCl, bafilomycin and NAC, suggesting that apoptotic cells negativelysignal the inflammasome pathway. To further illustrate the level ofapoptotic cells negative signaling cytochalasin D, a pharmacologic agentthat inhibits actin polymerization, has been used to prevent andeliminate engulfment. Using this approach, binding of apoptotic cells tomacrophages without engulfment was shown to be fully sufficient forinhibition of IL-1β secretion (FIG. 15B, *p<0.01, one way ANOVA).

Given the need for TLR triggering through NF-κB signaling, and the factthat apoptotic cells can inhibit NF-κB, and therefore inhibit IL-1βsecretion in the absence of inflammasome inhibition, a set ofexperiments was initiated to elucidate whether IL-1β secretion isinhibited both at NF-κB and NLRP3 levels by apoptotic cells. Residentperitoneal macrophages (pMΦ) were either incubated with apoptotic cells,washed, and primed with LPS following stimulation with variousinflammasome inducers, or were first primed with LPS, allowingaccumulation of de novo pro-IL-1β transcription and then treated withapoptotic cells and inducers.

Prior apoptotic cells treatment inhibited the secretion of activatedIL-1β at pre-transcription levels, attributing it to NF-κB pathwayinhibition. But more importantly, the inhibition effect e.g. IL-1βsecretion, was also observed after the accumulation of de novo IL-1βthat is, after LPS priming. This inhibition was obtained using threedifferent activators of the NLRP3 inflammasome triggering mechanisms;including nigericin, calcium pirophosphate (CPPD) and monosodium urate(MSU), suggesting a more robust inhibitory effect on NLRP3 inflammasome(FIG. 15C-E, p<0.001, one way ANOVA). Inhibition of secretion at posttranscription level were also acquire using cytochalasin D, whichfurther support inflammasome negative signaling upon recognition ofapoptotic cells without engulfment (data not shown).

The results were further verified by western blot analysis againstpro-IL-1β (p35) and cleaved and secreted IL-1β (p17). Macrophages (pMΦ)were incubated either in the presence of apoptotic cells for 2 hfollowed by LPS priming for 1 h (ApoCell delivered pre-transcription),or first primed with LPS (to promote NF-κB signaling) for 1 h and thenincubated with apoptotic cells for 2 h (ApoCell deliveredpost-transcription). The macrophages were then optionally incubated withan inflammasome inducer—either nigericin (2.5 μM) or calciumpyrophosphate dihydrate 200 μg/mL (CPPD).

Similarly to ELISA results, a diminished cleaved IL-1β subunit in thesupernatant of macrophages treated with LPS prior to apoptotic celltreatment or apoptotic cells prior to LPS was observed (FIG. 16A-B,IL-1β; upper panel). Of note, LPS priming by itself, leads toaccumulation of de novo pro-IL-1β in macrophages as can be seen in thecell lysate fraction but none in the supernatant (third lane from leftin FIGS. 16A-B, IL-1β; lower panel). The reduction in IL-1β levels wasseen even if NF-κB triggering with LPS was allowed before exposure toapoptotic cells. Comparable results are shown with caspase-1 where lessactivation of caspase-1 was measured at pre- and post-transcriptionlevels following apoptotic cells treatment in the presence of differentinducers.

Western blot analysis was also used to verify that apoptotic cells andLPS by themselves do not affect the secretion of mature IL-1β orcaspase-1 activation in the absence of inflammasome triggering. Indeed,no secretion of mature IL-1β or caspase-1 activation was observed, bothat the pre- and post-transcription levels, indicating the involvement ofNLRP3-inflammasome (FIG. 17A-B). Taken together, apoptotic cells appearto have a distinct inhibition effects on NF-κB and NLRP3.

Example 10 The Apoptotic Cell Anti-Inflammasome Effect is Mediated ViaROS, Lysosome Stabilization, and K⁺ Efflux

Activation of the NLRP3 inflammasome was suggested to be ROS-dependent,and indeed many NLRP3 stimulators also induce ROS generation. DSS wasalso found to generate ROS during NLRP3 activation and accumulation ofIL-1β.

The effect of apoptotic cell treatment on ROS generation was examined byboth fluorescent microscopy and real time flow cytometry. In agreementwith the previous observations, peritoneal macrophages incubated withDSS were found to induce ROS, similarly to pyocyanin, another inducer ofROS (FIG. 18). When macrophages were pretreated with apoptotic cells andthen treated with DSS, a marked and significant reduction in ROSgeneration was seen (FIG. 18 and FIG. 19, p<0.05, one way ANOVA). Thereduction is similar to the effect obtained by ROS inhibitorN-Acetyl-cysteine (NAC).

A second mechanism described as an important mechanism in NLRP3activation is lysosomal damage, leading to cytosolic release oflysosomal content that in turn triggers the inflammasome. It was alsosuggested that DSS triggers inflammasomes by lysosomal damage. To testwhether apoptotic cells prevent lysosomal damage, cytosolic staining wasperformed with acridine orange, a dye emitting green fluorescence whenmonomericly bonded to DNA and RNA and red fluorescence when dimerized inacidic compartments. The extent of the red fluorescence correlates withthe level of intracellular acidic lysosomes.

DSS treatment resulted in a significant decrease in red fluorescenceintensity (FIG. 20A, p<0.05, one way ANOVA), indicating lysosomaldamage, and in agreement with previous findings for DSS and crystals.When the macrophages were treated with apoptotic cells prior to the DSSchallenge, a significant increase in the number of acidic compartmentswas detected suggesting stabilization of the lysosomal compartment (FIG.20A, p<0.03, one way ANOVA). This observation was confirmed usingconfocal microscopy, which showed a more diffuse cytosolic stainingpattern when macrophages were treated with DSS, indicating rupture oflysosomes (data not shown). However, when treated with apoptotic cellsprior to DSS challenge, the lysosomes appeared intact. Taken together,this data suggests that lysosomal compartment stabilization is involvedin inflammasome regulation by apoptotic cells.

A third NLRP3 activation mechanism was suggested to involve changes inthe intracellular ionic milieu, either via ATP and the P2X₇ receptor orby pore forming toxins, and possibly also involving pannexin-1. BlockingK⁺ efflux or applying high concentrations of K⁺ prevented NLRP3inflammasome activation by many agents, including DSS.

To evaluate to role of K⁺ efflux in the presence of apoptotic cells, theeffect of macrophages pretreated with apoptotic cells on inhibitingIL-1β secretion following LPS and nigericin challenge was examined. Asseen in FIG. 20B, IL-1β secretion was indeed inhibited up to 70% byapoptotic cells in a dose-dependent manner, although less competent athigh concentrations.

Example 11 The ApoCell Cell Preparation Comprises Methylprednisolone

In order to determine the amount of methylprednisolone in the ApoCellfinal product, cell-preparations were produced according to theproduction method of the invention. During preparation, the cells wereincubated in an incubation medium comprising 50 μg/mL ofmethylprednisolone for six hours. At the end of apoptosis induction thecells are washed and re-suspended in PBS. Final volume of ApoCellproduct after collection of quality control samples was 300 ml. Theresidual amount of methyl prednisolone in the supernatant of the ApoCellfinal product was determined on final products prepared from three runs.Methylprednisolone levels were determined using reversed-phase liquidchromatography (HPLC). Assays were qualified and performed by SpectrolabAnalytical Laboratory, Rehovot, Israel. The levels of residual methylprednisolone in the ApoCell final product are presented in Table 3below.

The range of residual methylprednisolone concentration in the finalproduct is 3.7 mg/L in the lowest cell dose of ApoCell product and 21.9mg/L in the highest cell dose. The range of total methyl prednisolone infinal dose is 1.11-6.57 mg in correlation to the ApoCell dose. Theresults demonstrate that the amount of methylprednisolone present inApoCell, including the highest cohort, is negligible relative to thedose of methylprednisolone received by a patient as part of the generaltreatment protocol during Bone Marrow Transplantation.

TABLE 3 Residual Methylprednisolone in ApoCell Final Product Total Totalamount Residual number of of Methyl concentration of cells inprednisolone in Methyl Cohort No. ApoCell Run No. final doseprednisolone (cells/kg) dose (Batch ID No.) 1.11 mg 3.7 mg/L 1  2.45× 10⁹ Run 1 (3.5 × 10⁷ cells/kg) (Batch ID: 0021) 3.3 mg 11.2 mg/L 3   7 × 10⁹ Run 2 (1.4 × 10⁸ cells/kg) (Batch ID: 0024) 6.57 mg 21.9 mg/L4 11.34 × 10⁹ Run 3 (2.1 × 10⁸ cells/kg) (Batch ID: 0022)

Example 12 Effect of Plasma Comprising a High-Triglyceride Level onApoCell Cell-Preparation Yield

In order to determine the effect of plasma containing high levels oftriglycerides (TG) on the cell yield of the ApoCell composition, cellswere collected from a healthy donor with normal triglyceride levels andwere frozen in a freezing medium devoid of anticoagulant. Followingthawing, the cells were divided into four treatment groups:

-   -   (1) Incubation in incubation medium containing autologous plasma        with normal triglyceride levels.    -   (2) Incubation in incubation medium containing heterologous        plasma with normal triglyceride levels.    -   (3) Incubation in incubation medium containing heterologous        hyperlipidemic plasma with a TG level of 23.5 millimol/liter.    -   (4) Incubation in incubation medium containing heterologous        hyperlipidemic plasma with a TG level of 5.6 millimol/liter.

As can be seen in the results depicted in Table 4, incubation in thepresence of plasma containing a high triglyceride level resulted inlower ApoCell yield (An⁺=cells positive for Annexin V staining,PI⁻=cells negative for propidium iodide staining). Similarly, as can beseen in the results depicted in Table 5, preparing the cell-preparationof the invention from donors having high triglyceride levels using thesame method as in Examples 1-4 herein above, resulted in low ApoCellyield.

TABLE 4 ApoCell yield as function of plasma type ApoCell yield fromfrozen Apoptosis Necrosis Treatment sample (%) (An⁺ PI⁻ %) (PI %)Autologous plasma (normal 56.2 50 1.9 TG level) Heterologous plasma with52 57 2.6 normal TG level Heterologous plasma with TG 39.7 51 2.8 levelof 23.5 millimol/liter Heterologous plasma with TG 15.6 42 2.8 level of5.6 millimol/liter

TABLE 5 ApoCell yield as function of triglyceride level in donor's bloodTG final yield of ApoCell: Collected plasma actual cell number ×10⁹ cellnumber level Collection Donor and (% of frozen cells) (×10⁹) (mmol/l)ID# ID# 0.79 (4.95%) 16.1 6.6 406-2 406 2.04 (12.8%) 15.9 2.6 406-3

Example 13 Effect of Anticoagulant on ApoCell Cell-Preparation Yield inthe Presence of Plasma Comprising a High-Triglyceride Level

In order to determine whether addition of anticoagulant duringproduction of ApoCell results in a high and stable cell yield, cellswere collected from a healthy donor using leukapheresis and used toproduce the ApoCell product as described herein. During production, thecells were frozen in freezing media and incubated in incubation mediaboth comprising either:

-   1. Autologous plasma with normal triglyceride levels. No    anti-coagulant in freezing or incubation media.-   2. Heterologous plasma from a healthy donor with normal triglyceride    levels. No anti-coagulant in freezing or incubation media.-   3. Heterologous hyperlipidemic plasma with high triglyceride levels.    No anti-coagulant in freezing or incubation media.-   4. Heterologous hyperlipidemic plasma with high triglyceride levels    (same as in clause 3) and 5% of anticoagulant solution (ACD formula    A+10 U/ml heparin).

The cells from each treatment group were exposed to the same plasma thatthey were frozen with, throughout the experiment.

In agreement with the results presented in Example 11, the results inTable 6 demonstrate that high plasma level of triglycerides results in alow yield of the ApoCell cell-preparation, as can be seen in treatment 3(10.4% out of frozen cells). Unexpectedly, addition of anticoagulantduring the preparation process had a protective effect, thus enablingarrival at a normal cell-preparation yield, as can be seen in treatment4 (42.6% out of frozen cells).

TABLE 6 ApoCell yield summary in different treatments Yield at thawingYield of ApoCell Treatment Treatment (% from preparation # descriptionfrozen cells) (% from frozen cells) 1 Autologous plasma 82.1 49.5 2Heterologous plasma 76.6 50.1 3 Heterologous 11.3 10.4 plasma, high TG,no anticoagulant 4 Heterologous 69.9 42.6 plasma, high TG, withanticoagulant

Example 14 The Yield of ApoCell Cell-Preparation is Affected by Additionof Anticoagulant During Various Stages of the Preparation Process

In order to examine the effect of anticoagulant addition duringdifferent stages of ApoCell production on the cell-yield of the finalpreparation, cells were collected by leukapheresis from the same threehealthy donors (denoted 0036, 0037 and 0038) at two different medicalcenters (denoted medical centers 1 and 2). In addition, cells werecollected from two more healthy donors (denoted 0039-1 and 0040-1) atmedical center 1. The cell collection at the medical centers differed inthe protocol of anticoagulant addition during cell collection asfollows:

-   -   1. Center 1—5000 U of Heparin (Heparin sodium, Fresenius) is        injected into a bag of Acid Citrate Dextrose formula A (ACD        formula A); the heparin and ACD formula A circulate in the        leukapheresis machine such that a small fraction reaches the        donor and the collection bags (cells and plasma).    -   2. Center 2—5000 U of Heparin (Heparin sodium, Fresenius) is        injected directly into the cell collection bag, thus the Heparin        does not circulate within the leukapheresis machine, does not        reach the donor and does not reach the plasma collection bag.        ACD formula A, however, circulates in the machine and reaches        the donor and collection bags (cells and plasma).

Therefore, the main difference between the production method in centers1 and 2 is the concentration of Heparin in the collection bags. Theconcentration of Heparin in the cell collection bag in medical center 2is higher in comparison to the concentration of Heparin in the cellcollection bag in medical center 1. Additionally, the plasma collectionbag in medical center 2 is substantially devoid of heparin.

Following cell-collection at the two medical centers, ApoCell cellpreparations were produced from the collected cells under 4 conditions:

-   -   1. F⁻/Inc⁻=No anti-coagulant was added during freezing,        incubation or washing steps.    -   2. F⁻/Inc⁺=No anti-coagulant was added during freezing and        washing, anticoagulant was added during incubation.    -   3. F⁺/Inc⁺=Anti-coagulant was added during freezing, during        washing steps following freezing and during incubation.    -   4. F⁺/Inc⁻=Anti-coagulant was added during freezing and washing        steps following freezing but was not added during incubation.

Each freezing, incubation or washing media containing an anticoagulantduring this experiment, contained 5% anti-coagulant. The anti-coagulantused during the experiment was ACD Formula A supplemented with 10 U/mlheparin.

As can be seen in Table 7, the average yield of ApoCell cell-preparationproduced without addition of anti-coagulant during freezing orincubation was lower in medical center 2 than in medical center 1 (25.2vs. 51.4%, respectively). Addition of anti-coagulant during incubationor during both incubation and freezing resulted in a high and stablecell yield of above 40% in both medical centers 1 and 2. Therefore,addition of anti-coagulant during incubation or during both incubationand freezing results in a high yield of the ApoCell cell-preparation,regardless of the cell collection conditions. The yield values in Table7 refer to cells in the ApoCell composition out of frozen collectedcells from which the composition derived.

TABLE 7 ApoCell yield summary as a function of anticoagulant addition atdifferent stages in the manufacturing process in two medical centersYield ApoCell at preparation (% of frozen) Experimental group F⁺/Inc⁻F⁺/Inc⁺ F⁻/Inc⁺ F⁻/Inc⁻ Medical 2 1 2 1 2 1 2 1 Center 29.7 62 61.2 62.550.4 53.4 45.5 52.1 Donor 0036 (collections 0036-1 &SH0036-3) 23 63.5 5253.5 42 36.7 19 50.5 Donor 0037 (collections 0037-1 & SH0037-2) 1.7 58.447.8 53.6 35.4 42 11.2 42.7 Donor 0039 (collections 0038-1 &SH0038-2)36.2 34.1 52 57.6 Donor 0039 (collection 0039-1) 58.8 53.3 49 54.3 Donor0040 (collection 0040-1) 18.1 55.8 53.7 52.4 42.6 46.6 25.2 51.4 AVERAGE

Example 15 Characterization of Polymorphonuclear Cells within ApoCellCell Preparations

In order to evaluate the percentage of granulocytes within the ApoCellcell preparations, the percentage of polymorphonuclear cells wasmeasured within cells collected by leukapheresis and within the ApoCellcompositions which were produced from each collection (Col—the number ofthe cell collection examined from the same patient). As can be seen inTable 8, the percentage of granulocytes within the ApoCell compositionis much lower than the percentage of polymorphonuclear cells within theleukapheresis-collected mononuclear enriched cell fraction.

TABLE 8 ApoCell polymorphonuclear cell percentage At leukapheresis ofApoCell (final product) mononuclear enriched fraction Identity/Purity:CD15^(high) Identity/Purity: PMNs, by Cohort and by flow cytometrySysmex hematology analyzer patient (granulocytes, %) (%) number 0.4Col-1: 6.6 Cohort 1-1 0.21 Col-1: 5 Cohort 1-2 0.16 Col-1: 5.4 Cohort1-3 0.22 Col-1: 4.8 Cohort 2-1 0.58 Col-1: 31.7 Cohort 2-2 Col-2: 12.50.14 Col-1: 8.5 Cohort 2-3 0.11 Col-1: 14.9 Cohort 2-4 Col-2: 14.2Col-1: 10.2 Cohort 3-1 0.08 Col-2: 8 Col-3: 12.3 0.27 Col-1: 35 Cohort3-2 Col-2: 27.6 Col-3: 12.7 0.25 Col-1: 15.9 Cohort 3-3 Col-2: 15.9Col-3: 6 0.2 Col-1: 7.7 Cohort 4-1 Col-2: 5.7 0.17 Col-1: 16.2 Cohort4-2 Col-2: 6.2 Col-3: 10.7 0.08 Col-1: 8.5 Cohort 4-3 Col-2: 4.7 Col-3:7.9 0.22% (0.08-0.58%) 12.1% Average % (4.7-35%) (range)

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

The invention claimed is:
 1. A stable, high yield early-apoptoticmononuclear-enriched cell population comprising a yield of at least 30%early apoptotic mononuclear-enriched cells, wherein said early-apoptoticcell population is stable for more than 24 hours, wherein production ofsaid cell population comprises the following steps: (a) freezing amononuclear-enriched cell population in a freezing medium comprising ananticoagulant; (b) thawing said mononuclear-enriched cell population;and (c) inducing apoptosis in said thawed mononuclear-enriched cellpopulation, said inducing comprising incubating said population in amedium comprising methylprednisolone and an anticoagulant.
 2. The cellpopulation of claim 1, wherein said mononuclear enriched cells compriseat least one cell type selected from the group consisting oflymphocytes, monocytes and natural killer cells.
 3. The cell populationof claim 1, wherein said mononuclear enriched cells are collected byleukapheresis.
 4. The cell population of claim 1, wherein saidmononuclear enriched cells are obtained from a subject in need ofreceiving administration of a stable early apoptotic cell population. 5.The cell population of claim 1, wherein said mononuclear enriched cellsare obtained from a subject allogeneic with a subject in need ofreceiving administration of a stable early apoptotic cell population. 6.A pharmaceutical composition, comprising a stable early-apoptoticmononuclear-enriched cell population comprising at least 30% earlyapoptotic mononuclear-enriched cells wherein said early-apoptotic cellpopulation is stable for more than 24 hours, wherein production of saidcell population comprises the following steps: (a) freezing amononuclear-enriched cell population in a freezing medium comprising ananticoagulant; (b) thawing said mononuclear-enriched cell population;and (c) inducing apoptosis in said thawed mononuclear-enriched cellpopulation, said inducing comprising incubating said population in amedium comprising methylprednisolone and an anticoagulant.
 7. Thepharmaceutical composition of claim 6, wherein said composition furthercomprises an anti-coagulant.
 8. A method for producing a pharmaceuticalcomposition comprising a stable early-apoptotic mononuclear-enrichedcell population comprising at least 30% early apoptoticmononuclear-enriched cells wherein said early-apoptotic cell populationis stable for more than 24 hours, said method comprising a step offreezing a population of mononuclear-enriched cells in a freezing mediumcomprising an anticoagulant, and a step of inducing apoptosis of saidmononuclear-enriched population in a medium comprisingmethylprednisolone and an anticoagulant, or a combination thereof. 9.The method of claim 8, wherein said mononuclear enriched cells arecollected by leukapheresis.
 10. The method of claim 8, wherein saidmononuclear enriched cells are obtained from a subject in need ofreceiving administration of a stable early apoptotic cell population.11. The method of claim 8, wherein said mononuclear enriched cells areobtained from a subject allogeneic with a subject in need of receivingadministration of a stable early apoptotic cell population.